E-selectin compositions and use thereof for inducing e-selectin tolerance

ABSTRACT

The invention relates to compositions and methods for treating or preventing vascular dementia in a mammal comprising mucosal administration of an amount of E-selectin polypeptide sufficient to induce bystander immune tolerance in the mammal. Another aspect of the invention relates to compositions useful for treating or preventing vascular dementia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/072,914, filed Feb. 28, 2008, which is a continuation-in-part of PCTApplication No. PCT/US2006/034432, filed Aug. 30, 2006, which claims thebenefit of U.S. Provisional Application No. 60/712,359, filed Aug. 30,2005. U.S. application Ser. No. 12/072,914, filed Feb. 28, 2008, is alsoa continuation-in-part of PCT Application No. PCT/US2007/021682, filedOct. 9, 2007, which claims benefit of U.S. Provisional Application No.60/828,732, filed Oct. 9, 2006, and U.S. Provisional Application No.60/905,741, filed Mar. 8, 2007. U.S. application Ser. No. 12/072,914,filed Feb. 28, 2008, is also a continuation-in-part of U.S. applicationSer. No. 11/820,326, filed Jun. 19, 2007, which is a continuation ofU.S. application Ser. No. 10/296,423, filed Jun. 11, 2003, issued asU.S. Pat. No. 7,261,896 on Aug. 28, 2007, which is the U.S. NationalStage of International Application No. PCT/US01/16583, filed May 23,2001, published in English under PCT Article 21(2), which claims thebenefit of U.S. Provisional Application No. 60/206,693, filed May 24,2000. All of the above-listed applications are specifically incorporatedherein by reference in their entireties, and the benefit of theirearlier filing dates is claimed.

FIELD

The invention relates to compositions and methods for treatment ofvascular dementia in a mammalian subject that involve inducing toleranceto E-selectin in the subject.

BACKGROUND

Vascular dementia can be defined as the loss of cognitive functionresulting from ischemic, ischemic-hypoxic, or hemorrhagic brain lesionsas a result of cardiovascular diseases and cardiovascular pathologicchanges. See, e.g., G. C. Roman, Med. Clin. North. Am., 86, pp. 477-99(2002). Vascular dementia is a chronic disorder. The symptoms ofvascular dementia include cognitive loss, headaches, insomnia and memoryloss. Vascular dementia may be caused by multiple strokes (MID orpost-stroke dementia) but also by single strategic strokes, multiplelacunes, and hypoperfusion lesions such as border zone infarcts andischemic periventricular leukoencephalopathy (Binswanger's disease).See, G. C. Roman, supra. In Asian countries such as China, Japan andKorea, vascular dementia is observed in over 60% of patients withdementia. Primary and secondary prevention of stroke and cardiovasculardisease decreases the burden of vascular dementia.

Treatment of vascular dementia typically involves control of riskfactors (i.e., hypertension, diabetes, smoking, hyperfibrinogenemia,hyperhomocystinemia, orthostatic hypotension, cardiac arrhythmias). See,G. C. Roman, supra. Researchers have also investigated whether hormonereplacement therapy and estrogen replacement therapy could delay theonset of dementia in women. See, E. Hogervorst et al., Cochrane DatabaseSyst. Rev., 3, CD003799 (2002). However, such hormone replacementtherapy has negative side effects. Moreover, although aspirin is widelyprescribed for vascular dementia, there is very limited evidence thataspirin is actually effective in treating vascular dementia patients.See, P. S. Williams et al., Cochrane Database Syst. Rev., 2, CD001296(2000). Nimodipine has been implicated as a drug demonstratingshort-term benefits in vascular dementia patients, but has not beenjustified as a long-term anti-dementia drug. See, J. M. Lopez-Arrietaand J. Birks, Cochrane Database Syst. Rev., 3, CD000147 (2002). Inaddition, clinical efficacy data of piracetam does not support the useof this drug in the treatment of dementia or cognitive impairment. L.Flicker and G. Grimley Evans, Cochrane Database Syst. Rev., 2, CD001011(2001).

Thus, new agents and procedures for treating vascular dementia areneeded.

SUMMARY

The invention involves methods and compositions for preventing andtreating vascular dementia. Surprisingly, the inventors have discoveredthat vascular dementia can be treated by inducing immunologicaltolerance to E-selectin, a cell adhesion molecule that mediates theadhesion of various leukocytes, including neutrophils, monocytes,eosinophils, natural killer (NK) cells, and a subset of T cells, toactivated endothelium. Such immunological tolerance leads to the releaseimmune system suppressive cytokines after subsequent stimulation byE-selectin, which is released in response to endothelia activation.

Thus, one aspect of the invention is a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and an effective amountof E-selectin, wherein the formulation is formulated for mucosaladministration of E-selectin. For example, the mucosal administrationcan be intranasal, oral, enteral, vaginal, rectal, or respiratoryadministration. In some embodiments, the formulation is formulated forintranasal administration, for example, as an aerosol. The aerosol canbe a dry aerosol. Alternatively, the aerosol can be an atomized aqueoussolution. The E-selectin is an E-selectin polypeptide. Such anE-selectin polypeptide can be a mammalian E-selectin polypeptide, forexample, a human E-selectin, a bovine E-selectin, a murine E-selectin, arat E-selectin or any other E-selectin polypeptide from a mammaliansource.

The pharmaceutical formulation of the invention is typicallyadministered in an effective amount (i.e., a therapeutically effectiveamount). Such an effective amount of E-selectin is generally sufficientto induce tolerance to E-selectin in a mammal. In some embodiments, aneffective amount of E-selectin is sufficient to promote bystander-effecttolerance to E-selectin in a mammal. Examples of effective amounts ofE-selectin include ranges of E-selectin of about 0.005 mg to about 500mg. Another example of an effective amount of E-selectin is a range ofE-selectin of about 5 μg to about 50 mg.

Another aspect of the invention is a method for treating or preventingvascular dementia in a mammal comprising mucosal administration of anamount of E-selectin polypeptide sufficient to induce bystander immunetolerance in the mammal. Such vascular dementia can involve reducedblood flow to the brain. In some embodiments, the E-selectin isadministered to mucosal surfaces of the mammal. For example, suchmucosal administration of E-selectin can include nasal, oral, enteral,vaginal, rectal, or respiratory administration. In some embodiments, theadministration is nasal or intranasal. The inventive methods can involvea series of separate E-selectin administrations. In some embodiments,the method involves a first series of administrations of E-selectin overa period of about two weeks. Such a first series of administrations caninclude about three to about seven administrations of E-selectin overthe period of about two weeks. The method can further comprise at leastone booster series of administrations of E-selectin after at least twoweeks from the first series of administrations. In some embodiments,each booster series of administrations comprise about three to aboutseven administrations of E-selectin over the period of two weeks.

Another aspect of the invention is a method for treating or preventingvascular dementia in a mammal comprising mucosal administration of anamount of E-selectin polypeptide sufficient to induce bystander immunetolerance in the mammal.

The E-selectin employed in the methods and compositions of the inventioncan include any of the following sequences: SEQ ID NO: 5-8, 18, 19,30-33, or any combination thereof.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B is the mucosal tolerance induction schedules employed forillustrative experiments described herein. For induction of mucosaltolerance, intranasal application of E-selectin was carried out. FIG. 1Ashows the schedule for rats that received just a single series ofE-selectin or PBS (control) administrations; rats receiving the singleseries of administrations are sometimes referred to herein as the“non-booster group.” FIG. 1B shows the schedule for rats that received aseries of booster administrations of E-selectin or PBS (control) everythree weeks; animals receiving such a series of administrations aresometimes referred to herein as the “booster group.”

FIG. 2 graphically illustrates the delayed type hypersensitivity (DTH)reaction in E-selectin-tolerized rats compared with PBS-tolerized rats.For this experiment, rats received an intranasal administration ofE-selectin or phosphate buffered saline (PBS, control), and then wereimmunized with E-selectin in the footpad prior to challenge by aninjection of E-selectin in the ear. This bar graph illustrates thechange in thickness of the ear in animals that received intranasalinstillation of E-selectin compared to the ear swelling in animals thatreceived intranasal PBS. E-selectin administration on a singletolerization schedule significantly suppressed the delayed typehypersensitivity (DTH) induction of ear swelling in these animals.Therefore, intranasal instillation of E-selectin in the doses used inthese animals does produce a state of immunological tolerization.

FIG. 3 graphically illustrates the discrimination indices of E-selectintolerized and non-tolerized rats for the object recognition test. Thediscrimination indices of the E-selectin and sham groups weresignificantly increased as compared with the PBS group. *: p<0.05, **:p<0.01, ***: p<0.001 by Fisher's protected least significant differenceprocedure, as compared to PBS-treated animals.

FIG. 4 graphically illustrates of the percent of alternation by rats onthe T-maze spontaneous alternation test. Rats have an instinctivebehavioral tendency to alternate their choices between the arms of theT-maze more often than they repeat their initial choice. In theE-selectin-treated animals, the percent of rats that alternated wassignificantly increased at 90 days, as compared with the PBS-treatedanimals. *: p<0.05 by χ² test.

FIG. 5A-C graphically illustrates the percentages of correct armentrance on the T maze left/right discrimination memory retention test.FIG. 5A shows the percentage of correct choices made by E-selectintolerized, PBS control and sham operated animals two weeks after carotidartery ligation. FIG. 5B shows the percentage of correct choices made byE-selectin tolerized, PBS control and sham operated animals six weeksafter carotid artery ligation. FIG. 5C shows the percentage of correctchoices made by E-selectin tolerized, PBS control and sham operatedanimals ten weeks after carotid artery ligation. Some variability in theE-selectin group responses was observed at six weeks after ligation(FIG. 5B), but in general, by the third day after acquisition, thepercentages of correct T-arm entries of the E-selectin and sham-operatedanimals were significantly increased as compared with the PBS group.

FIG. 6A-I shows photomicrographs of luxol fast blue stained sections ofthe corpus callosum (A, B, C), caudoputamen (D, E, F) and optic nerve(G, H, I) from rats that were subjected to a sham operation (C, F, I) orto bilateral ligation of the carotid arteries in animals that alsoreceived intranasal PBS (A, D, G) or E-selectin (B, E, H) on a boostertolerization schedule. Note that the extent of the white matterrarefaction was less severe in the E-selectin treated and sham-operatedrats as compared with PBS group.

FIG. 7A-F shows photomicrographs of sections immunohistochemical stainedusing a cocktail of monoclonal antibodies directed againstnon-phosphorylated neurofilaments (SMI 311) that were obtained from thecorpus callosum (A, B, C) and caudoputamen (D, E, F) from rats. The ratswere subjected to a sham operation (C, F) or to bilateral ligation ofthe carotid arteries in animals that also received intranasal PBS (A, D)or E-selectin (B, E) on a booster tolerization schedule. Note that theextent of the white matter rarefaction was less severe in the E-selectintreated and sham-operated rats as compared with PBS group.

FIG. 8 graphically illustrates the fiber densities of the corpuscallosum, caudoputamen and optic nerve in rats subjected to a shamoperation or to bilateral ligation of the carotid arteries andintranasal administration of either PBS or E-selectin on a boostertolerization schedule. The fiber densities in E-selectin-treated animalswere significantly higher than those in PBS-treated animals.

FIG. 9A-F shows photomicrographs of sections immunohistochemicallystained for MHC class II antigens from the corpus callosum of rats thatwere subjected to a sham operation (C, F) or of rats that were subjectedto bilateral ligation of the carotid arteries and intranasal PBS (A, D)or E-selectin (B, E) on a booster tolerization schedule. InE-selectin-treated rats, the number of microglia/macrophages positivelyimmuno-labeled for MHC class II antigen in the white matter lesions weresomewhat reduced in comparison to PBS-treated animals.

FIG. 10 shows histograms of the numerical densities of MHC class IIimmunopositive microglia/macrophages in the corpus callosum of ratssubjected to a sham operation, or to bilateral ligation of the carotidarteries in animals that also received intranasal PBS or E-selectin on abooster tolerization schedule.

FIG. 11A-B illustrates that CD4 positive T cells infiltrate braintissues after carotid artery occlusion. FIG. 11A-B show rat corpuscallosum sections immunohistochemically stained for CD4 (a marker for Tcells). Sham-treated rats exhibited little or no CD4 positive T cellinfiltration (FIG. 11A). In contrast, after carotid artery occlusion,increased numbers of CD4 positive T cells were observed in the corpuscallosum of rats (FIG. 11B).

FIG. 12A-F shows photomicrographs of immunohistochemically stainedsections for detection of TNF-α in the corpus callosum. The rats weresubjected to a sham operation (C, F) or to bilateral ligation of thecarotid arteries in animals that also received intranasal PBS (A, D) orE-selectin (B, E) on a booster tolerization schedule. In theE-selectin-treated and sham-operated animals, TNF-immunopositive vesselswere markedly less prominent than in PBS-treated animals.

FIG. 13 shows histograms of the numerical density ofTNF-α-immunopositive vessels in the corpus callosum of rats subjected toa sham operation, or to bilateral ligation of the carotid arteries inanimals that also received intranasal PBS or E-selectin on a boostertolerization schedule. In the E-selectin-treated and sham-operatedanimals, the number of TNF-α-immunopositive vessels was significantlyreduced as compared with the PBS-treated animals.

FIG. 14A-F shows photomicrographs of sections immunohistochemicallystained for detection of E-selectin in the corpus callosum. The ratswere subjected to a sham operation (C, F) or to bilateral ligation ofthe carotid arteries in animals that also received intranasal PBS (A, D)or E-selectin (B, E) on a booster tolerization schedule. The sectionswere taken 90 days after carotid ligation. In the E-selectin-treated andsham-operated animals, E-selectin-immunopositive vessels were lessprominent as compared to the PBS-treated animals.

FIG. 15 shows histograms of the numerical density ofE-selectin-immunopositive vessels in the corpus callosum of ratssubjected to a sham operation, or to bilateral ligation of the carotidarteries and either intranasal PBS or intranasal E-selectin on a boostertolerization schedule. In the E-selectin-treated and sham-operatedanimals, the number of E-selectin-immunopositive vessels wassignificantly reduced as compared with the PBS-treated animals.

FIG. 16 illustrates that occlusion of blood vessels feeding braintissues leads to a number of problems, including disturbances in axonaltransport, demyelination, induction of metalloproteinases (MMPs), bloodbrain barrier problems, activation of glial cells, infiltration oflymphocytes, edema, inflammation and immunological reactions that alllead to heightened tissue damage and further vascular injury.

FIG. 17A-C provides a comparison of E-selectin sequences where Line 1 iswild type E-Selectin (human), GenBank Acc. No. M30640 (SEQ ID NO: 21);Line 2: wild type E-Selectin (human), GenBank Acc. No. NM_(—)000450 (SEQID NO: 22); Line 3: “new” recombinant E-Selectin, no tags (SEQ ID NO:23). Line 4: “old” recombinant E-Selectin protein with c-myc, Histidinetags (SEQ ID NO: 24). The Underlined sequences are signal peptidesequences; the symbol ****** indicates that the sequences are part ofthe transmembrane domain; the symbol ###### indicates that the sequencesare c-myc and/or Histidine tags; the symbol ////// indicates that thesequences Lectin C-type domain sequences; the symbol @ @ @ @ @ @indicates that the sequences are Calcium binding EGF-like domainsequences. See, Nession et al., PNAS 87, 1673-1677 (1990); Zhang et al.,FEMS Microbiol Lett 227, 303-309 (2003); Kiely et al., J Immunol 171,3216-3224 (2003).

FIG. 18 is a comparison of human (SEQ ID NO: 25, top sequence) and mouse(SEQ ID NO: 26, bottom sequence) E-selectin sequences.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file, created on Jul.27, 2010, 104 KB, which is incorporated by reference herein.

DETAILED DESCRIPTION

The invention provides compositions and methods for treating, inhibitingand/or preventing negative consequences of vascular dementia.

DEFINITIONS

As used herein, “tolerance” refers to an antigen-induced immuneunresponsiveness upon re-exposure to the antigen. The antigen haspreviously been administered to induce such immune unresponsiveness. Theinduced immune unresponsiveness may be specific for the administeredantigen or may be antigen-non-specific as a result of production of anantigen-non-specific suppressor substance such as transforming growthfactor beta (TGF-β), interleukin-4 (IL-4) or interleukin-10 (IL-10).

As used herein “bystander tolerance” means that T-cells, which areprimed to recognize a specific antigen (E-selectin), release immunesystem suppressive cytokines after subsequent stimulation by thatantigen (E-selectin). Such suppressor T cells arise in the mucosalimmune system and migrate to systemic sites where, upon antigen-specificreactivation, the suppressor T cells release TGF-β, IL-4, IL-10 andother suppressive cytokines

A delayed type hypersensitivity reaction as used herein is a measure ofwhether the immune system actively reacts to an antigen or whether theimmune system exhibits tolerance towards the antigen. An antigen isintroduced intradermally, and after about 48-72 hours post-injection thesite of intradermal administration is observed. If the immune systemdoes not exhibit tolerance, the injection site will appear red,inflamed, thickened, and tender. The swelling and thickening of the skinare a result of an immune response. The lack of a delayed typehypersensitivity response to the antigen indicates that the immunesystem is tolerant of the antigen.

As used herein, a “subject” is a mammal or bird to which the E-selectincompositions of the invention are administered. Thus, the subject can bebovine, rat, mouse, dog, pig, horse, goat, monkey, ape, human or otherdomestic or zoo mammal. In addition, the subject can be chicken, turkey,parrot or other domestic or zoo bird.

As used herein, vascular dementia is a loss of cognitive function as aresult of diminished blood flow to the brain. Vascular dementia canarise from diminished blood flow in arteries within the heart and/or inblood vessels leading to the brain. While vascular dementia need not bea result of blockage in blood vessels within the brain, in someinstances, vascular dementia can occur as a result of such diminishedblood flow in blood vessels within the brain. Vascular dementia canoccur as a result of single event that reduces blood flow from the heartand/or with blood vessels leading to the brain. However, vasculardementia can also have a slow onset, for example, as a result ofprogressive decrease in blood flow from the heart to the brain overtime.

E-Selectin

E-selectin (also known as ELAM-1, CD62, and CD62E) is acytokine-inducible cell surface glycoprotein that is found onendothelial cells. E-selectin is a cell adhesion molecule that mediatesthe adhesion of various leukocytes, including neutrophils, monocytes,eosinophils, natural killer (NK) cells, and a subset of T cells, toactivated endothelium. See, e.g., Bevilacqua, et al., “Endothelialleukocyte adhesion molecule 1: an inducible receptor for neutrophilsrelated to complement regulatory proteins and lectins,” Science 243;1160 (1989); Graber, et al., “T cells bind to cytokine-activatedendothelial cells via a novel, inducible sialoglycoprotein andendothelial leukocyte adhesion molecule-1” J. Immunol. 145: 819 (1990);Carlos, et al., “Human monocytes bind to two cytokine-induced adhesiveligands on cultured human endothelial cells: endothelial-leukocyteadhesion molecule-1 and vascular cell adhesion molecule-1” Blood 77:2266 (1991); Hakkert, et al., “Neutrophil and monocyte adherence to andmigration across monolayers of cytokine-activated endothelial cells: thecontribution of CD18, ELAM-1, and VLA-4” Blood 78: 2721 (1991); andPicker, et al., “ELAM-1 is an adhesion molecule for skin-homing T cells”Nature 349: 796 (1991).

E-selectin is expressed in vascular endothelial tissue. Pober, J. S., etal., J. Immunol. 136: 1680 (1986); Bevilacqua M. P., et al., Proc. Natl.Acad. Sci. 84: 9238 (1987). Expression of E-selectin is induced inresponse to the cytokines IL-1 and TNF, as well as bacteriallipopolysaccharide (LPS), through transcriptional up-regulation. Poberet al., supra; see also, Montgomery, et al., “Activation ofendothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription”Proc. Natl. Acad. Sci. 88: 6523 (1991)). Some workers hypothesize thatactivation of vascular endothelial cells is involved in inflammatoryvascular tissue damage leading to thrombosis. Fareed, J. et al.,“Molecular markers of hemostatic activation. Implications in thediagnosis of thrombosis, vascular, and cardiovascular disorders,” Clin.Lab. Med. 15: 39 (1995).

Structurally, E-selectin belongs to a family of adhesion moleculestermed “selectins.” This family also includes P-selectin and L-selectin.Review articles relating to these selectins are provided in Lasky,“Selectins: interpreters of cell-specific carbohydrate informationduring inflammation” Science 258: 964 (1992) and Bevilacqua and Nelson,“Selectins” J. Clin. Invest. 91: 379 (1993). These molecules arecharacterized by common structural features such as an amino-terminallectin-like domain, an epidermal growth factor (EGF) domain, and adiscrete number of complement repeat modules (approximately 60 aminoacids each) similar to those found in certain complement bindingproteins.

Examples of nucleic acid and amino acid sequences for different typesand species of E-selectin can be found in the art, for example, in theNCBI database. See website at ncbi.nlm.nih.gov. Thus, for example, theNCBI database provides a human E-selectin precursor amino acid sequenceas accession number P16581 (gi: 126180). This sequence is provided belowfor easy reference as SEQ ID NO1.

  1 MIASQFLSAL TLVLLIKESG AWSYNTSTEA MTYDEASAYC 41 QQRYTHLVAI QNKEEIEYLN SILSYSPSYY WIGIRKVNNV 81 WVWVGTQKPL TEEAKNWAPG EPNNRQKDED CVEIYIKREK121 DVGMWNDERC SKKKLALCYT AACTNTSCSG HGECVETINN161 YTCKCDPGFS GLKCEQIVNC TALESPEHGS LVCSHPLGNF201 SYNSSCSISC DRGYLPSSME TMQCMSSGEW SAPIPACNVV241 ECDAVTNPAN GFVECFQNPG SFPWNTTCTF DCEEGFELMG281 AQSLQCTSSG NWDNEKPTCK AVTCRAVRQP QNGSVRCSHS321 PAGEFTFKSS CNFTCEEGFM LQGPAQVECT TQGQWTQQIP361 VCEAFQCTAL SNPERGYMNC LPSASGSFRY GSSCEFSCEQ401 GFVLKGSKRL QCGPTGEWDN EKPTCEAVRC DAVHQPPKGL441 VRCAHSPIGE FTYKSSCAFS CEEGFELHGS TQLECTSQGQ481 WTEEVPSCQV VKCSSLAVPG KINMSCSGEP VFGTVCKFAC521 PEGWTLNGSA ARTCGATGHW SGLLPTCEAP TESNIPLVAG561 LSAAGLSLLT LAPFLLWLRK CLRKAKKFVP ASSCQSLESD 601 GSYQKPSYILThe mature sequence for this human E-selectin extends from about aminoacid 22 to amino acid 610. The sequence for this mature E-selectinpolypeptide is therefore as follows (SEQ ID NO: 2).

 22                        WSYNTSTEA MTYDEASAYC 41 QQRYTHLVAI QNKEEIEYLN SILSYSPSYY WIGIRKVNNV 81 WVWVGTQKPL TEEAKNWAPG EPNNRQKDED CVEIYIKREK121 DVGMWNDERC SKKKLALCYT AACTNTSCSG HGECVETINN161 YTCKCDPGFS GLKCEQIVNC TALESPEHGS LVCSHPLGNF201 SYNSSCSISC DRGYLPSSME TMQCMSSGEW SAPIPACNVV241 ECDAVTNPAN GFVECFQNPG SFPWNTTCTF DCEEGFELMG281 AQSLQCTSSG NWDNEKPTCK AVTCRAVRQP QNGSVRCSHS321 PAGEFTFKSS CNFTCEEGFM LQGPAQVECT TQGQWTQQIP361 VCEAFQCTAL SNPERGYMNC LPSASGSFRY GSSCEFSCEQ401 GFVLKGSKRL QCGPTGEWDN EKPTCEAVRC DAVHQPPKGL441 VRCAHSPIGE FTYKSSCAFS CEEGFELHGS TQLECTSQGQ481 WTEEVPSCQV VKCSSLAVPG KINMSCSGEP VFGTVCKFAC521 PEGWTLNGSA ARTCGATGHW SGLLPTCEAP TESNIPLVAG561 LSAAGLSLLT LAPFLLWLRK CLRKAKKFVP ASSCQSLESD 601 GSYQKPSYILAn extracellular E-selectin domain may be used for tolerization of asubject. The extracellular domain of the human E-selectin provided aboveincludes a sequence of about amino acid 22 to about amino acid 556 andtherefore has the following sequence (SEQ ID NO: 3).

 22                        WSYNTSTEA MTYDEASAYC 41 QQRYTHLVAI QNKEEIEYLN SILSYSPSYY WIGIRKVNNV 81 WVWVGTQKPL TEEAKNWAPG EPNNRQKDED CVEIYIKREK121 DVGMWNDERC SKKKLALCYT AACTNTSCSG HGECVETINN161 YTCKCDPGFS GLKCEQIVNC TALESPEHGS LVCSHPLGNF201 SYNSSCSISC DRGYLPSSME TMQCMSSGEW SAPIPACNVV241 ECDAVTNPAN GFVECFQNPG SFPWNTTCTF DCEEGFELMG281 AQSLQCTSSG NWDNEKPTCK AVTCRAVRQP QNGSVRCSHS321 PAGEFTFKSS CNFTCEEGFM LQGPAQVECT TQGQWTQQIP361 VCEAFQCTAL SNPERGYMNC LPSASGSFRY GSSCEFSCEQ401 GFVLKGSKRL QCGPTGEWDN EKPTCEAVRC DAVHQPPKGL441 VRCAHSPIGE FTYKSSCAFS CEEGFELHGS TQLECTSQGQ481 WTEEVPSCQV VKCSSLAVPG KINMSCSGEP VFGTVCKFAC521 PEGWTLNGSA ARTCGATGHW SGLLPTCEAP TESNIP

In some embodiments human E-selectin may be administered to a subject.As is known to the skilled artisan, some sequence variation exists inhuman E-selectins. Thus, other human E-selectin amino acid sequences canbe found in the NCBI database, for example, as accession numbersAANO1237 (gi: 22536178), CAA17434 (gi: 3115964), AAA52376 (gi: 537524),CAI19357 (gi: 56417699), among others. According to the invention, anysuch human E-selectin polypeptides can be used for administration to asubject.

As indicated above, wild type E-selectins have a total about of 589amino acids. Such wild type E-selectins include a lectin domain, anepidermal growth factor-like (EGF) domain, and a series of between 2 and9 consensus repeat domains similar to those of complement proteins.Thus, wild type E-selectin, for example, the E-selectin sequencesprovided in FIG. 17, can generally include the structural elements shownbelow.

Amino acids 1-21: signal sequence

Amino acids 22-140: lectin like domain

Amino acid 144-175: EGF like domain

Amino acid 180-237: first consensus repeat domain

Amino acid 242-300: second consensus repeat domain

Amino acid 300-363: third consensus repeat domain

Amino acid 367-426: fourth consensus repeat domain

Amino acid 430-489: fifth consensus repeat domain

Amino acid 493-548: sixth consensus repeat domain

A membrane spanning domain of about 22 amino acids and an intracellulardomain of about 32 amino acids are also present at the carboxyl terminusof wild type E-selectin (see FIG. 17). However, neither themembrane-spanning domain nor the intracellular domain need be present inthe E-selectins used in the compositions and methods of the invention.Moreover, several of the consensus repeat domains can be eliminated fromthe E-selectin used in the compositions and methods of the invention.

Thus, in some embodiments, the E-selectin is a soluble E-selectin thatdoes not contain the membrane spanning domain or the intracellulardomain. Soluble E-selectin can be generated by enzymatic cleavage (toeliminate the membrane spanning domain and/or the intracellular domain)or by recombinant expression of the soluble E-selectin portion of themolecule. The exact amino acid sequence of E-selectin can therefore varydepending on the cleavage site chosen for deleting the membrane spanningand/or the intracellular domains, or the C-terminus selected for makinga recombinant soluble E-selectin. In addition, the number ofcomplement-like consensus repeats can vary.

Thus, in some embodiments, the extracellular portion of the E-selectinmolecule is used. Such an extracellular region of E-selectin can have upto about 550 amino acids or more desirably up to about 535 amino acids.However, in many embodiments the extracellular domain of E-selectin hasless than about 550 to 535 amino acids. For example, the extracellulardomain used in the compositions and methods of the invention can haveabout 1 to about 260 amino acids, or any integer in between, fewer aminoacids than the 535-550 amino acids that generally comprises theE-selectin extracellular domain. Thus, the extracellular domain ofE-selectin that is used in the compositions and methods of the inventioncan have at least about 275, about 280, about 285, about 290, about 295,about 300, about 310, about 315, about 320, about 325 amino acids or anyinteger from at least about 275 to at least about 325 amino acids.

In general, the extracellular domain of E-selectin includes, from theamino terminus of the E-selectin protein: the lectin domain, theepidermal growth factor-like (EGF) domain, and a series of between 2 and9 consensus repeat domains similar to those of complement proteins.Thus, the E-selectin can have about 2, about 3, about 4, about 5, about6, about 7, about 8 or about 9 consensus repeat domains. Depending onthe number of consensus repeat domains, the total number of amino acidsand the molecular weight of E-selectin will therefore change.

The consensus repeat domains of E-selectin are also called complementcontrol protein (CCP) modules, short consensus repeats (SCRs) or SUSHIrepeats. These consensus repeat domains contain approximately 60 aminoacid residues and have been identified in several proteins of thecomplement system. For example, there are two consensus repeat domainsat positions 13-53 and 57-112 in the following sequence (NCBI accessionnumber AAQ67702; gi: 34420911; SEQ ID NO: 4).

  1 PKGLVRCAHS PIGEFTYKSS CAFSCEEGFE LYGSTQLECT 41 SQGQWTEEVP SCQVVKCSSL AVPGKINMSC SGEPVFGTVC 81 KFACPEGWTL NGSAARTCGA TGHWSGLLPT CEAPTESNIP 121 LVAGLSAAGL SLLTLAPFIn one embodiment, a human E-selectin protein is used in thecompositions and methods of the invention that has about 306 amino acids(e.g. SEQ ID NO: 5).

  1 MPLYKLLNVL WLVAVSNAIP GSWSYNTSTE AMTYDEASAY 41 CQQRYTHLVA IQNKEEIEYL NSILSYSPSY YWIGIRKVNN 81 VWVWVGTQKP LTEEAKNWAP GEPNNRQKDE DCVEIYIKRE121 KDVGMWNDER CSKKKLALCY TAACTNTSCS GHGECVETIN161 NYTCKCDPGF SGLKCEQIVN CTALESPEHG SLVCSHPLGN201 FSYNSSCSIS CDRGYLPSSM ETMQCMSSGE WSAPIPACNV241 VECDAVTNPA NGFVECFQNP GSFPWNTTCT FDCEEGFELM281 GAQSLQCTSS GNWDNEKPTC KAVTRSThe SEQ ID NO: 5 E-selectin sequence is part of the third sequenceidentified as the “new” recombinant E-Selectin with no tags shown inFIG. 18. In another embodiment, the human E-selectin protein used in thecompositions and methods of the invention that has about 304 amino acids(e.g. SEQ ID NO: 6), because the C-terminal arginine and serine residuesare not present.

  1 MPLYKLLNVL WLVAVSNAIP GSWSYNTSTE AMTYDEASAY 41 CQQRYTHLVA IQNKEEIEYL NSILSYSPSY YWIGIRKVNN 81 VWVWVGTQKP LTEEAKNWAP GEPNNRQKDE DCVEIYIKRE121 KDVGMWNDER CSKKKLALCY TAACTNTSCS GHGECVETIN161 NYTCKCDPGF SGLKCEQIVN CTALESPEHG SLVCSHPLGN201 FSYNSSCSIS CDRGYLPSSM ETMQCMSSGE WSAPIPACNV241 VECDAVTNPA NGFVECFQNP GSFPWNTTCT FDCEEGFELM281 GAQSLQCTSS GNWDNEKPTC KAVT

In another embodiment, a human E-selectin protein without a signalsequence is used in the compositions and methods of the invention thathas about 284 amino acids (e.g. SEQ ID NO: 7).

  1 WSYNTSTEAM TYDEASAYCQ QRYTHLVAIQ NKEEIEYLNS 41 ILSYSPSYYW IGIRKVNNVW VWVGTQKPLT EEAKNWAPGE 81 PNNRQKDEDC VEIYIKREKD VGMWNDERCS KKKLALCYTA121 ACTNTSCSGH GECVETINNY TCKCDPGFSG LKCEQIVNCT161 ALESPEHGSL VCSHPLGNFS YNSSCSISCD RGYLPSSMET201 MQCMSSGEWS APIPACNVVE CDAVTNPANG FVECFQNPGS241 FPWNTTCTFD CEEGFELMGA QSLQCTSSGN WDNEKPTCKA 281 VTRS

In a further embodiment, the human E-selectin protein used in thecompositions and methods of the invention that has about 282 amino acids(e.g. SEQ ID NO: 8), because the signal sequence and the C-terminalarginine and serine residues are not present.

  1 WSYNTSTEAM TYDEASAYCQ QRYTHLVAIQ NKEEIEYLNS 41 ILSYSPSYYW IGIRKVNNVW VWVGTQKPLT EEAKNWAPGE 81 PNNRQKDEDC VEIYIKREKD VGMWNDERCS KKKLALCYTA121 ACTNTSCSGH GECVETINNY TCKCDPGFSG LKCEQIVNCT161 ALESPEHGSL VCSHPLGNFS YNSSCSISCD RGYLPSSMET201 MQCMSSGEWS APIPACNVVE CDAVTNPANG FVECFQNPGS241 FPWNTTCTFD CEEGFELMGA QSLQCTSSGN WDNEKPTCKA 281 VTThese approximate 282-284 amino acid sequences for E-selectin has thelectin domain, the EGF domain, and two complement-like consensusrepeats.

In some embodiments, a signal sequence may be present on the N-terminusof the E-selectin. One example of a signal sequence that can be used isthe MGWSWIFLFLLSGTASVHS (SEQ ID NO: 27) signal sequence. Another exampleof a signal sequence that can be used is the MPLYKLLNVLWLVAVSNAI (SEQ IDNO: 28) signal sequence. Also in some embodiments, a C-terminal tagsequence may be used with the E-selectin. One example of a C-terminaltag sequence that can be used is a histidine tag sequence, for example,the GGASTRAAEQKLI SEEDLNGTRSGHHHHHH (SEQ ID NO: 29) tag sequence.

In addition, in some embodiments it may be useful to administerE-selectin from non-human species to the subject. Thus, for example,non-human E-selectin may optimally inhibit inflammation and/or inducetolerization to E-selectin in some human subjects. Therefore, theinvention is directed to administering non-human E-selectin to subjects,and such non-human E-selectin can include just the extracellular portionof the E-selectin and/or the extracellular portion of E-selectin withjust 2 to about 9 consensus repeat domains. Many sources and examples ofnon-human E-selectin are available. For example, nucleic acid and aminoacid sequences for different types of non-human E-selectin can be foundin the art, for example, in the NCBI database. See website atncbi.nlm.nih.gov. Thus, for example, bovine, rat, mouse, dog, pig,horse, goat, monkey, ape or other mammalian E-selectin polypeptides canbe administered to a subject. Sequences for such mammalian E-selectinsare available, for example, in the NCBI database.

One example of a bovine E-selectin polypeptide sequence that can befound in the NCBI database is the bovine E-selectin sequence withaccession number P98107 (gi: 1346435). This bovine E-selectin sequenceis the precursor sequence and is provided below for easy reference (SEQID NO: 9).

  1 MIVSQYLSAL TFVLLLFKES RTWSYHASTE MMTFEEARDY 41 CQKTYTALVA IQNQEEIEYL NSTFSYSPSY YWIGIRKING 81 TWTWIGTNKS LTKEATNWAP GEPNNKQSDE DCVEIYIKRE121 KDSGKWNDEK CTKQKLALCY KAACNPTPCG SHGECVETIN161 NYTCQCHPGF KGLKCEQVVT CPAQKHPEHG HLVCNPLGKF201 TYNSSCSISC AEGYLPSSTE ATRCMSSGEW STPLPKCNVV241 KCDALSNLDN GVVNCSPNHG SLPWNTTCTF ECQEGYKLTG281 PQHLQCTSSG IWDNKQPTCK AVSCAAISHP QNGTVNCSHS321 VVGDFAFKSS CHFTCAEGFT LQGPTQVECT AQGQWTQRVP361 VCEVVRCSRL DVSGKLNMNC SGEPVLGTEC TFACPERWTL401 NGSVVLTCGA TGHWSGMLPT CEAPTVSQTP LAVGLSTAGV441 SLVTIPSFLF WLLKRLQKKA KKFSPASSCS SLKSNGCYST 481 PSKLIThe mature sequence for this bovine E-selectin extends from about aminoacid 23 to amino acid 485. The sequence for this mature bovineE-selectin polypeptide is therefore as follows (SEQ ID NO: 10).

 23                         WSYHASTE MMTFEEARDY 41 CQKTYTALVA IQNQEEIEYL NSTFSYSPSY YWIGIRKING 81 TWTWIGTNKS LTKEATNWAP GEPNNKQSDE DCVEIYIKRE121 KDSGKWNDEK CTKQKLALCY KAACNPTPCG SHGECVETIN161 NYTCQCHPGF KGLKCEQVVT CPAQKHPEHG HLVCNPLGKF201 TYNSSCSISC AEGYLPSSTE ATRCMSSGEW STPLPKCNVV241 KCDALSNLDN GVVNCSPNHG SLPWNTTCTF ECQEGYKLTG281 PQHLQCTSSG IWDNKQPTCK AVSCAAISHP QNGTVNCSHS321 VVGDFAFKSS CHFTCAEGFT LQGPTQVECT AQGQWTQRVP361 VCEVVRCSRL DVSGKLNMNC SGEPVLGTEC TFACPERWTL401 NGSVVLTCGA TGHWSGMLPT CEAPTVSQTP LAVGLSTAGV441 SLVTIPSFLF WLLKRLQKKA KKFSPASSCS SLKSNGCYST 481 PSKLIAn extracellular E-selectin domain may be used for tolerization of asubject. The extracellular domain of the bovine E-selectin providedabove includes a sequence of about amino acid 23 to about amino acid 430and therefore has the following sequence (SEQ ID NO: 11).

 23                         WSYHASTE MMTFEEARDY 41 CQKTYTALV AIQNQEEIEYL NSTFSYSPSY YWIGIRKING 81 TWTWIGTNK SLTKEATNWAP GEPNNKQSDE DCVEIYIKRE121 KDSGKWNDE KCTKQKLALCY KAACNPTPCG SHGECVETIN161 NYTCQCHPG FKGLKCEQVVT CPAQKHPEHG HLVCNPLGKF201 TYNSSCSIS CAEGYLPSSTE ATRCMSSGEW STPLPKCNVV241 KCDALSNLD NGVVNCSPNHG SLPWNTTCTF ECQEGYKLTG281 PQHLQCTSS GIWDNKQPTCK AVSCAAISHP QNGTVNCSHS321 VVGDFAFKS SCHFTCAEGFT LQGPTQVECT AQGQWTQRVP361 VCEVVRCSR LDVSGKLNMNC SGEPVLGTEC TFACPERWTL401 NGSVVLTCG ATGHWSGMLPT CEAPTVSQTP

As is known to the skilled artisan, some sequence variation exists amongbovine E-selectins. Thus, other bovine E-selectin amino acid sequencescan be found in the NCBI database, for example, as accession numbersS36772 (gi: 480377) and NP 776606 (gi: 27806407), among others.According to the invention, any such bovine E-selectin polypeptides canbe used for tolerization of a subject to E-selectin.

One example of a rat E-selectin polypeptide sequence that can be foundin the NCBI database is the rat E-selectin sequence with accessionnumber P98105 (gi: 1346437). This rat E-selectin sequence is theprecursor sequence and is provided below for easy reference (SEQ ID NO:12).

  1 MNASCFLSAL TFVLLIGKSI AWYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN STLRYSPSYY WIGIRKVNNV 81 WIWVGTQKPL TEEAKNWAPG EPNNKQRNED CVEIYIQRPK121 DSGMWNDERC DKKKLALCYT ASCTNTSCSG HGECVETINS161 YTCKCHPGFL GPKCDQVVTC QEQEYPDHGS LNCTHPFGLF201 SYNSSCSFSC ERGYVPSSME TTVRCTSSGE WSAPAPACHV241 VECKALTQPA HGVRKCSSNP GSYPWNTTCT FDCEEGYRRV281 GAQNLQCTSS GVWDNEKPSC KAVTCDAIPR PQNGSVSCSN321 STAGALAFKS SCNFTCEHSF TLQGPAQVEC SAQGQWTPQI361 PVCKASQCEA LSAPQRGHMK CLPSASAPFQ SGSSCKFSCD401 EGFELKGSRR LQCGPRGEWD SEKPTCAGVQ CSSLDLPGKM441 NMSCSGPAVF GTVCEFTCPE GWTLNGSSIL TCGATGRWSA481 MLPTCEAPAN PPRPLVVALS VAATSLLTLS SLIYVLKRFF521 WKKAKKFVPA SSCQSLQSFE NYQGPSYIIThe mature sequence for this rat E-selectin extends from about aminoacid 22 to amino acid 549. The sequence for this mature rat E-selectinpolypeptide is therefore as follows (SEQ ID NO: 13).

 22                        WYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN STLRYSPSYY WIGIRKVNNV 81 WIWVGTQKPL TEEAKNWAPG EPNNKQRNED CVEIYIQRPK121 DSGMWNDERC DKKKLALCYT ASCTNTSCSG HGECVETINS161 YTCKCHPGFL GPKCDQVVTC QEQEYPDHGS LNCTHPFGLF201 SYNSSCSFSC ERGYVPSSME TTVRCTSSGE WSAPAPACHV241 VECKALTQPA HGVRKCSSNP GSYPWNTTCT FDCEEGYRRV281 GAQNLQCTSS GVWDNEKPSC KAVTCDAIPR PQNGSVSCSN321 STAGALAFKS SCNFTCEHSF TLQGPAQVEC SAQGQWTPQI361 PVCKASQCEA LSAPQRGHMK CLPSASAPFQ SGSSCKFSCD401 EGFELKGSRR LQCGPRGEWD SEKPTCAGVQ CSSLDLPGKM441 NMSCSGPAVF GTVCEFTCPE GWTLNGSSIL TCGATGRWSA481 MLPTCEAPAN PPRPLVVALS VAATSLLTLS SLIYVLKRFF521 WKKAKKFVPA SSCQSLQSFE NYQGPSYIIAn extracellular E-selectin domain may be used for tolerization of asubject. The extracellular domain of the rat E-selectin provided aboveincludes a sequence of about amino acid 22 to about amino acid 494 andtherefore has the following sequence (SEQ ID NO: 14).

 21                       AWYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN STLRYSPSYY WIGIRKVNNV 81 WIWVGTQKPL TEEAKNWAPG EPNNKQRNED CVEIYIQRPK121 DSGMWNDERC DKKKLALCYT ASCTNTSCSG HGECVETINS161 YTCKCHPGFL GPKCDQVVTC QEQEYPDHGS LNCTHPFGLF201 SYNSSCSFSC ERGYVPSSME TTVRCTSSGE WSAPAPACHV241 VECKALTQPA HGVRKCSSNP GSYPWNTTCT FDCEEGYRRV281 GAQNLQCTSS GVWDNEKPSC KAVTCDAIPR PQNGSVSCSN321 STAGALAFKS SCNFTCEHSF TLQGPAQVEC SAQGQWTPQI361 PVCKASQCEA LSAPQRGHMK CLPSASAPFQ SGSSCKFSCD401 EGFELKGSRR LQCGPRGEWD SEKPTCAGVQ CSSLDLPGKM441 NMSCSGPAVF GTVCEFTCPE GWTLNGSSIL TCGATGRWSA 481 MLPTCEAPAN PPRP

One example of a mouse E-selectin polypeptide sequence that can be foundin the NCBI database is the mouse E-selectin sequence with accessionnumber B42755 (gi: 25295806). This mouse E-selectin sequence is theprecursor sequence and is provided below for easy reference (SEQ ID NO:15).

  1 MNASRFLSAL VFVLLAGEST AWYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN SNLKHSPSYY WIGIRKVNNV 81 WIWVGTGKPL TEEAQNWAPG EPNNKQRNED CVEIYIQRTK121 DSGMWNDERC NKKKLALCYT ASCTNASCSG HGECIETINS161 YTCKCHPGFL GPNCEQAVTC KPQEHPDYGS LNCSHPFGPF201 SYNSSCSFGC KRGYLPSSME TTVRCTSSGE WSAPAPACHV241 VECEALTHPA HGIRKCSSNP GSYPWNTTCT FDCVEGYRRV281 GAQNLQCTSS GIWDNETPSC KAVTCDAIPQ PQNGFVSCSH321 STAGELAFKS SCNFTCEQSF TLQGPAQVEC SAQGQWTPQI361 PVCKAVQCEA LSAPQQGNMK CLPSASGPFQ NGSSCEFSCE401 EGFELKGSRR LQCGPRGEWD SKKPTCSAVK CDDVPRPQNG441 VMECAHATTG EFTYKSSCAF QCNEGFSLHG SAQLECTSQG481 KWTQEVPSCQ VVQCPSLDVP GKMNMSCSGT AVFGTVCEFT521 CPDDWTLNGS AVLTCGATGR WSGMPPTCEA PVSPTRPLVV561 ALSAAGTSLL TSSSLLYLLM RYFRKKAKKF VPASSCQSLQ 601 SFENYHVPSY NVThe mature sequence for this mouse E-selectin extends from about aminoacid 22 to amino acid 612. The sequence for this mature mouse E-selectinpolypeptide is therefore as follows (SEQ ID NO: 16).

 22                        WYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN SNLKHSPSYY WIGIRKVNNV 81 WIWVGTGKPL TEEAQNWAPG EPNNKQRNED CVEIYIQRTK121 DSGMWNDERC NKKKLALCYT ASCTNASCSG HGECIETINS161 YTCKCHPGFL GPNCEQAVTC KPQEHPDYGS LNCSHPFGPF201 SYNSSCSFGC KRGYLPSSME TTVRCTSSGE WSAPAPACHV241 VECEALTHPA HGIRKCSSNP GSYPWNTTCT FDCVEGYRRV281 GAQNLQCTSS GIWDNETPSC KAVTCDAIPQ PQNGFVSCSH321 STAGELAFKS SCNFTCEQSF TLQGPAQVEC SAQGQWTPQI361 PVCKAVQCEA LSAPQQGNMK CLPSASGPFQ NGSSCEFSCE401 EGFELKGSRR LQCGPRGEWD SKKPTCSAVK CDDVPRPQNG441 VMECAHATTG EFTYKSSCAF QCNEGFSLHG SAQLECTSQG481 KWTQEVPSCQ VVQCPSLDVP GKMNMSCSGT AVFGTVCEFT521 CPDDWTLNGS AVLTCGATGR WSGMPPTCEA PVSPTRPLVV561 ALSAAGTSLL TSSSLLYLLM RYFRKKAKKF VPASSCQSLQ 601 SFENYHVPSY NVAn extracellular E-selectin domain may be used for tolerization of asubject. The extracellular domain of the mouse E-selectin provided aboveincludes a sequence of about amino acid 22 to about amino acid 557 andtherefore has the following sequence (SEQ ID NO: 17).

 22                        WYYNASSEL MTYDEASAYC 41 QRDYTHLVAI QNKEEINYLN SNLKHSPSYY WIGIRKVNNV 81 WIWVGTGKPL TEEAQNWAPG EPNNKQRNED CVEIYIQRTK121 DSGMWNDERC NKKKLALCYT ASCTNASCSG HGECIETINS161 YTCKCHPGFL GPNCEQAVTC KPQEHPDYGS LNCSHPFGPF201 SYNSSCSFGC KRGYLPSSME TTVRCTSSGE WSAPAPACHV241 VECEALTHPA HGIRKCSSNP GSYPWNTTCT FDCVEGYRRV281 GAQNLQCTSS GIWDNETPSC KAVTCDAIPQ PQNGFVSCSH321 STAGELAFKS SCNFTCEQSF TLQGPAQVEC SAQGQWTPQI361 PVCKAVQCEA LSAPQQGNMK CLPSASGPFQ NGSSCEFSCE401 EGFELKGSRR LQCGPRGEWD SKKPTCSAVK CDDVPRPQNG441 VMECAHATTG EFTYKSSCAF QCNEGFSLHG SAQLECTSQG481 KWTQEVPSCQ VVQCPSLDVP GKMNMSCSGT AVFGTVCEFT521 CPDDWTLNGS AVLTCGATGR WSGMPPTCEA PVSPTRP

Another example of a mouse E-selectin polypeptide sequence that can befound in the NCBI database is the mouse E-selectin sequence withaccession number NP_(—)035475.1 (gi: 6755452). This mouse E-selectinsequence has the signal sequence and is provided below for easyreference (SEQ ID NO: 18).

  1 MGWSWIFLFL LSGTASVHSW YYNASSELMT YDEASAYCQR 41 DYTHLVAIQN KEEINYLNSN LKHSPSYYWI GIRKVNNVWI 81 WVGTGKPLTE EAQNWAPGEP NNKQRNEDCV EIYIQRTKDS121 GMWNDERCNK KKLALCYTAS CTNASCSGHG ECIETINSYT161 CKCHPGFLGP NCEQAVTCKP QEHPDYGSLN CSHPFGPFSY201 NSSCSFGCKR GYLPSSMETT VRCTSSGEWS APAPACHVVE241 CEALTHPAHG IRKCSSNPGS YPWNTTCTFD CVEGYRRVGA281 QNLQCTSSGI WDNETPSCKA VT

When the SEQ ID NO: 18 mouse E-selectin sequence does not have thesignal sequence, it has the following sequence (SEQ ID NO: 19).

  1                     W YYNASSELMT YDEASAYCQR 41 DYTHLVAIQN KEEINYLNSN LKHSPSYYWI GIRKVNNVWI 81 WVGTGKPLTE EAQNWAPGEP NNKQRNEDCV EIYIQRTKDS121 GMWNDERCNK KKLALCYTAS CTNASCSGHG ECIETINSYT161 CKCHPGFLGP NCEQAVTCKP QEHPDYGSLN CSHPFGPFSY201 NSSCSFGCKR GYLPSSMETT VRCTSSGEWS APAPACHVVE241 CEALTHPAHG IRKCSSNPGS YPWNTTCTFD CVEGYRRVGA281 QNLQCTSSGI WDNETPSCKA VT

Sources of E-selectin that can be used with the current inventioninclude E-selectin that has been substantially purified from naturalsources, recombinant E-selectin produced in prokaryotic or eukaryotichost cells by methods available in the art, and fragments of E-selectin.Furthermore, small organic molecules or peptides with structures thatmimic an immunoreactive portion of E-selectin can also be used.

In some embodiments, the E-selectin is produced by recombinantprocedures. For example, a codon-optimized nucleic acid encoding themouse E-selectin polypeptide with SEQ ID NO: 18, with the followingsequence (SEQ ID NO: 20) can be used for recombinant production of mouseE-selectin.

  1 ATGGGTTGGT CCTGGATCTT CCTGTTTCTC TTGTCTGGCA 41 CCGCTAGCGT GCACTCATGG TACTATAACG CCTCGAGTGA 81 GCTTATGACT TACGACGAAG CGTCCGCATA CTGCCAGCGT121 GATTATACAC ATCTGGTCGC TATTCAAAAT AAGGAGGAAA161 TCAACTACCT CAATTCTAAC TTGAAACACA GCCCCTCATA201 CTATTGGATT GGAATCCGCA AGGTTAACAA TGTATGGATC241 TGGGTGGGTA CGGGCAAACC TCTTACCGAG GAAGCCCAGA281 ACTGGGCGCC AGGAGAGCCG AACAATAAGC AAAGGAACGA321 AGATTGTGTC GAGATTTACA TCCAGAGAAC TAAGGATTCG361 GGTATGTGGA ACGACGAACG ATGCAATAAA AAGAAGCTGG401 CACTCTGTTA CACAGCTAGT TGCACGAACG CCTCCTGTTC441 TGGCCATGGA GAGTGCATTG AGACCATCAA CAGCTATACT481 TGCAAATGTC ACCCCGGTTT CTTGGGCCCT AATTGCGAAC521 AAGCTGTTAC ATGTAAGCCA CAGGAGCACC CGGATTACGG561 ATCACTGAAC TGCTCCCATC CCTTCGGTCC TTTTTCGTAC601 AATAGTTCTT GCAGCTTCGG CTGTAAACGT GGATATCTTC641 CATCATCCAT GGAAACCACG GTACGCTGCA CTTCGAGTGG681 TGAGTGGTCT GCGCCGGCCC CCGCATGTCA CGTGGTCGAA721 TGCGAGGCTC TCACCCATCC TGCCCACGGC ATCAGGAAGT761 GCAGCTCCAA CCCAGGATCA TACCCCTGGA ACACAACTTG801 TACCTTCGAC TGCGTTGAAG GTTACAGACG TGTGGGCGCG841 CAAAATTTGC AGTGTACGTC GTCTGGAATT TGGGACAACG881 AGACACCTAG TTGCAAGGCT GTCACTTAA

Recombinant procedures for production of E-selectin polypeptides canemploy expression systems for small or large scale production ofE-selectin. Expression systems useful for making E-selectin include, butare not limited to, cells or microorganisms that are transformed with arecombinant nucleic acid construct that contains a nucleic acid segmentencoding an E-selectin polypeptide. Examples of recombinant nucleic acidconstructs may include bacteriophage DNA, plasmid DNA, cosmid DNA, orviral expression vectors. Examples of cells and microorganisms that maybe transformed include bacteria (for example, E. coli or B. subtilis);yeast (for example, Saccharomyces and Pichia); insect cell systems (forexample, baculovirus in Spodoptera frugiperda, Sf9 cells); plant cellsystems; or mammalian cell systems (for example, COS, CHO, BHK, 293,VERO, HeLa, MDCK, W138, and NIH 3T3 cells). Also useful as host cellsare primary or secondary cells obtained directly from a mammal that aretransfected with a plasmid vector or infected with a viral vector.Examples of suitable expression vectors include, without limitation,plasmids and viral vectors such as herpes viruses, retroviruses,vaccinia viruses, attenuated vaccinia viruses, canary pox viruses,adenoviruses, adeno-associated viruses, lentiviruses and herpes viruses,among others. Synthetic methods may also be used to produce polypeptidesand peptide fragments of the invention. Such methods are known and havebeen reported. Merrifield, Science 85:2149 (1963).

In some embodiments, the expression system includes use of ChineseHamster Ovary (CHO) cells or insect cells as the host cells. Theglycosylation with a mammalian cell such as a CHO cell is known todiffer from that of an insect expression system such as the baculovirusexpression vector system. The difference is that glycosylation of aprotein molecule derived from the baculovirus vector inserted into aninsect expression system leads to an asparagine attacheddi-N-acetylglycosamine to which a terminal trimannose is attached. Thisis termed the paucimannose structure and it facilitates interaction withmannose receptors on antigen-presenting cells. Hence, there may be anadvantage in some situations to utilize a baculovirus expression vectorsystem. In other embodiments, a mammalian expression system may be used,where additional N-linked glycans may be attached to the three mannosesof the terminal trimannose (paucimannose) structure generated in theinsect expression system. These N-linked glycans includeN-acetylglycosamine, galactose, and N-acetylneuraminic acid (also knownas sialic acid). Therefore, a variety of host cells can be used togenerate E-selectin polypeptides with somewhat different glycosylationpatterns. The invention is directed to compositions and methods of usingE-selectin with any type of glycosylation, or no glycosylation.

Immune Tolerance

The immune system has the remarkable ability to mount a highly specificresponse against invading pathogens while ignoring self molecules. Thisspecificity is determined in part by the T lymphocyte, which expresses arandomly generated and unique T-cell receptor (TCR) that recognizes apeptide antigen bound to a major histocompatibility complex (MHC)molecule. MHC molecules can bind both to self peptides as well as toforeign peptides, where the self peptides are from the same organism asthe MHC molecules (i.e., the host) and the foreign peptides are from adifferent, foreign organism. Thus, the specificities of the peripheralTCR repertoire and/or the function of self-reactive T cells must beregulated such that the immune system ignores the self peptides orresponds in a way that does not injure the host. The physicalelimination of autoreactive T cells during thymocyte development is theprimary mechanism used by the immune system to establish suchself-tolerance. However, not all self peptides are present in thethymus. Therefore, the immune system must either ignore atissue-specific self peptide, or develop an active self-tolerance thatrelies on the suppression, physical elimination, or functionalinactivation of mature autoreactive T cells.

The following observations are generally applicable to immune tolerance:(1) tolerance refers to a selective inability of the immune system torespond to antigens and, for purposes of this invention, is a “learned”phenomenon; (2) both foreign and self-antigens can be targets oftolerogenic processes; (3) although tolerance can be mediated bysuppressor cells, tolerance is not the same as immune suppression,either mechanistically or clinically; (4) tolerance can be maintained byactive or passive processes and can result from cell inactivation,altered cellular function, or cell death; and (5) tolerance can beinduced centrally (in the thymus) or peripherally.

According to the invention, immune tolerance is generated by exposure ofmucosal surfaces to a tolerizing antigen (here, E-selectin). Immuneresponses in mucosal tissues are self-limited, and repeated challengewith selected antigens results in a diminished response. Mucosaladministration of both high- and low-dose antigen results in immunetolerance, in which the immune response to subsequent systemicadministration of antigen is blocked. However, at least two mechanismsof immune tolerance may exist. Tolerance to high-doses of an antigenappears to occur by inactivation or clonal deletion of Th1 and Th2cells. In contrast, tolerance to low doses of antigen leads to“bystander” immune suppression mediated by stimulation of regulatorycells to produce Th2- and Th3 type cytokines, with interleukin-4 (IL-4),interleukin-10 (IL-10) and TGF-β being the major suppressive cytokines.

Inactivation of T cells by the clonal deletion tolerance mechanism iscalled clonal anergy and was originally described using a tissue culturesystem of cloned T cells. Clonal anergy has since been defined as areversible, induced tolerance state in which the T lymphocyte cannotproduce its autocrine growth factor IL-2 or proliferate in response tothe antigen it recognizes. In vitro, this unresponsive state is inducedby stimulation of the T cell through its TCR in the absence ofcostimulatory signals, such as those occurring as a result of theinteraction of B7 molecules on the antigen presenting cell (APC) withCD28 receptors on the T cell. In the absence of such costimulatorysignals, T cells fail to proliferate, and TCR occupancy unaccompanied byproliferation down-regulates the T cell's responsiveness.

Bystander suppression relies on the induction of regulatory cells inmucosal tissues that are specific for the mucosally administeredantigen. So called “bystander antigens” cause regulatory (suppressor)T-cells to be induced in the gut-associated lymphoid tissue (GALT), orbronchial associated lymphoid tissue (BALT), or most generally, mucosaassociated lymphoid tissue (MALT). MALT includes both GALT and BALT.After migration to the diseased or affected organ, these regulatorycells can be activated by the presence of the antigen, and will secreteimmunosuppressive cytokines (IL-4, IL-10, and TGF-β), thereby leading tosuppression of ongoing immune responses to the antigen against whichtolerance was induced and to unrelated self antigens. Evidence suggeststhat immune regulation and bystander suppression occur afteradministration of intermediate or lower antigen doses, whereas clonaldeletion or clonal anergy of antigen-reactive lymphocytes generallyoccurs at high dosages.

IL-4, IL-10 and TGF-β are antigen-nonspecific immunosuppressive factorsthat suppress immune attack regardless of the antigen that triggers theattack. However, because oral or mucosal tolerization with a bystanderantigen only causes the release of these cytokines in the vicinity ofautoimmune attack, no systemic immunosuppression ensues. TGF-β isthought to be one of the most important cytokines for bystandertolerance. IL-4 enhances Th2 response (i.e., acts on T-cell precursorsand causes them to differentiate preferentially into Th2 cells at theexpense of Th1 responses). IL-4 also indirectly inhibits Th1exacerbation. IL-10 is a direct inhibitor of Th1 responses.

After orally tolerizing mammals afflicted with autoimmune diseaseconditions with bystander antigens, increased levels of TGF-β, IL-4, andIL-10 are observed at the locus of autoimmune attack (Chen, Y. et al.,“Regulatory T cell clones induced by oral tolerance: suppression ofautoimmune encephalomyelitis,” Science, 265: 1237-1240, (1994)). Thebystander suppression mechanism has also been confirmed by von Herrathet al., “Oral insulin treatment suppresses virus-inducedantigen-specific destruction of beta cells and prevents autoimmunediabetes in transgenic mice,” J. Clin. Invest., 96: 1324-1331, (1996).

According to the invention, inducing E-selectin tolerance has manyutilities. For example, it can be used in preventing and treatingvascular dementia, strokes and other forms of vascular disease.Additionally, it can be used in treating disorders in which E-selectinhas been determined, or may be determined, to play a role, such as, forexample, lung injury, psoriasis, contact dermatitis, inflammatory boweldisease, arthritis, and the like. (See, e.g., Washington R., et al.,“Expression of immunologically relevant endothelial cell activationantigens on isolated central nervous system microvessels from patientswith multiple sclerosis,” Ann. Neurol. 35: 89 (1994); Bevilacqua (1989);Bevilacqua and Nelson, “Selectins,” J. Clin. Invest. 91: 379 (1993);Koch, et al., “Immunolocalization of endothelial and leukocyte adhesionmolecules in human rheumatoid and osteoarthritic synovial tissues,” LabInvest. 64: 313 (1991); Mulligan, et al., “Role of endothelial-leukocyteadhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury inrats,” J. Clin. Invest. 88: 1396 (1991); and Mulligan, et al.,“Protective effects of oligosaccharides in P-selectin-dependent lunginjury,” Nature 364: 149 (1993)).

Vascular Dementia

As indicated above, vascular dementia is a loss of cognitive function asa result of diminished blood flow to the brain. Vascular dementia canarise from diminished blood flow in arteries within the heart and/or inblood vessels leading to the brain. Thus, while vascular dementia can bea result of blockage in blood vessels within the brain, it can also becaused by poor blood flow to the brain. Moreover, while vasculardementia may occur as a result of single event that reduces blood flowfrom the heart and/or with blood vessels leading to the brain, vasculardementia can also have a slow onset, for example, as a result ofprogressive decrease in blood flow from the heart to the brain overtime.

Vascular dementia can therefore result from ischemic or hemorrhagicbrain lesions as well as from lesions that develop elsewhere duringprotracted hypoperfusion. The subcortical ischemic form of vasculardementia is a common type of vascular cognitive impairment and dementia,and one of the major causes of cognitive decline in elderly people.Subcortical ischemic vascular dementia mainly results from small-vesseldisease, which causes lacunes and extensive white matter lesions, andcan be compared to large vessel dementia or cortical vascular dementia(Roman G C, Neurology. 1993; 43:250-260, Roman G C Lancet Neurol. 2002;1:426-436). The ischemic lesions in subcortical ischemic vasculardementia particularly affect the frontal-subcortical circuits, anobservation that explains the major cognitive and clinical neurologicaleffects of vascular dementia (Ishii N, Neurology 1986; 36: 340-45,Cummings J L, Arch Neurol 1993; 50:873-80). Subcortical ischemicvascular dementia is also caused by persistent hypertension (de Leeuw FE, Brain. 2002; 125:765-772) and hypoperfusion due to congestive heartfailure (Roman G C. Neurol Res. 2004; 26:454-458), atrial fibrillation(de Leeuw F E, Neurology. 2000; 54:1795-1801), and obstructive sleepapnea (Kamba M, J. Neurol. Neurosurg. Psychiatry. 2001; 71; 334-339).

Ischemic white matter lesions, a common finding in elderly people, arethe characteristic pathological changes in subcortical ischemic vasculardementia and cognitive impairment, and cognitive dysfunctions arerelated to lesion severity (Hachinski V C, Arch Neurol. 1987; 4:21-23,Pantoni L, Alzheimer Dis. Assoc. Disord. 1999; 13(suppl 3):S49-S54, deGroot J C, Neurology 2001; 56:1539-1545). Cerebrovascular white matterlesions constitute the core pathology in several types of vasculardementia, such as Binswanger's disease, cerebral amyloid angiopathy, andcerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL). These cerebrovascular white matterlesions are caused by chronic cerebral hypoperfusion, which result fromthe severe stenosis of several arteries or arterioles mainly in deepwhite matter (Pantoni L, Stroke 1997; 28:652-659, de Groot J C,Neurology 2001; 56:1539-1545, Roman G C, Neurol. Res. 2004; 26:454-458,Capizzano A A, Am J Neuroradiol 2000; 21:621-630).

Animal models exist for vascular dementia, permitting analysis of theeffects of drugs and drug dosages on the development, prognosis andrecovery from vascular dementia. In particular, cerebrovascular whitematter lesions can be experimentally induced in the rat brains as aresult of protracted hypoperfusion induced by the permanent occlusion ofboth common carotid arteries (Wakita H, Acta Neuropathol. (Berl) 1994;87: 484-492). In this model, cerebral blood flow decreases to about 40%of the normal blood flow and the gradually increase to about 82% ofnormal blood flow over extended periods of time (Tsuchiya M, Exp. BrainRes. 89:87-92 (1992): Otori T, Cerebrovasc. Dis. 6 (suppl): 71 (1996);Tomimoto H, Brain Nerve 49:639-644 (1997); Ouchi Y, J Nucl Med.39:198-202 (1998)). These animals exhibit delayed white matter lesionsand memory impairment correlated with the damage of frontal-subcorticalcircuits. This method of inducing forebrain ischemia can thus be used asa model for vascular dementia (Wakita H, Acta Neuropathol. (Berl) 87:484-492 (1994); Pappas B A, Brain Res. 708:50-58 (1996); Ohta H,Neuroscience 79:1039-1050 (1997); Wakita H, Brain Res. 924:63-70(2002)); Sarti C, Behav Brain Res. 136:13-20 (2002)).

Previous studies using this animal model for vascular dementia havedemonstrated that CD4- or CD8-positive T cells infiltrate in the neuralparenchyma, and that microglia, the immune effector cells of the centralnervous system, are activated and express MHC class I and II antigensbriefly after ischemia in a manner predictive of the extent and theseverity of demyelination and axonal damage (Wakita H, Acta Neuropathol.(Berl) 87: 484-492 (1994); Wakita H, Stroke 26:1415-1422 (1995); WakitaH, Brain Res. 792:105-113 (1998); Wakita H, Neuroreport 14:1461-1465(1999); Wakita H, Brain Res. 924:63-70 (2002)). The suppression of theseactivated microglia by immunosuppressive and anti-inflammatory drugsresults in an attenuation of the white matter lesions (Wakita H, Stroke26:1415-1422 (1995); Wakita H, Brain Res. 792:105-113 (1998); Wakita H,Neuroreport 14:1461-1465 (1999); Wakita H., Brain Res. 992:53-59(2003)). The activation of microglia can also be detected in the earlystage of human cerebrovascular white matter lesions, and is associatedwith degradation of myelin and axonal damage (Suenaga T, ActaNeuropathol (Berl). 87:450-455 (1994); Akiguchi I, Stroke. 28:1423-1429(1997)). These data suggest that the immunological and inflammatoryreactions can augment the white matter damage under chronic ischemia.

As described above, E-selectin, a glycoprotein, is a cell surface-boundleukocyte adhesion molecule specific to endothelial cells (Bevilacqua MP, Science 243(4895):1160-1165 (1989)). It mediates the interactionbetween leukocytes, platelets, and the endothelium (Bevilacqua (1989)).Normal resting endothelial cells do not express E-selectin (Pigott R,BBRC 187:584-9 (1992)). The expression of E-selectin is induced inresponse to proinflammatory cytokines, such as IL-1 and TNF, and itsincreased surface expression is a reflection of endothelial activation(Bevilcqua M P, Annu. Rev. Immunol. 11:767-804 (1993)). In patients withcerebrovascular disease, including subcortical ischemic vasculardementia, the serum concentration of the soluble isoform of E-selectinis increased (Fassbender K, Stroke 26:1361-1364 (1995); Frijns C J,Stroke 28: 2214-2218 (1997); Fassbender K, Stroke 30:1647-1650 (1999)).The upregulation of E-selectin expression in the ischemic cerebralvasculature has been shown in experimental cerebral ischemia (Wang X,Stroke 26:1665-1669 (1995); Haring H—P, Stroke 27:1386-1392 (1996);Zhang R L, J Cereb Blood Flow Metab. 16:1126-113 (1996); Huang J, Stroke31:3047-3053 (2000)). Moreover, administration of anti-E-selectinantibody reduces the infarct volume and neurological deficits in murinetransient focal ischemia model (Huang J, Stroke 31:3047-3053 (2000)).

In view of these observations, and the results provided herein, vesselactivation and E-selectin expression play a pivotal role in theinflammatory process and subsequent tissue injury after cerebralischemia through leukocyte-endothelial attachment and infiltration ofleukocytes.

Thus, a novel method to induce generation of regulatory T cells targetedto activating blood vessels has been developed involving administrationof E-selectin to induce mucosal tolerance to that antigen. Mucosaltolerance to E-selectin prevents ischemic and hemorrhagic strokes inspontaneously hypertensive stroke prone rats (Takeda H, Stroke33:2156-2164 (2002)) and protects against ischemic brain damage afterpermanent middle cerebral artery occlusion in spontaneously hypertensivestroke prone rats (Chen Y, Proc. Natl. Acad. Sci. U.S.A. 100:15107-12(2003)). These findings suggest that E-selectin participates ininflammation and immunological responses during and after an ischemicinsult and serves to target immunomodulatory regulatory T cells to bloodvessel segments that are undergoing endothelial cell activation. Asillustrated in a previous application by the inventors U.S. Ser. No.10/296,423 (filed Jun. 11, 2003, and incorporated herein in itsentirety), these regulatory T cells may prevent stroke and protectagainst ischemic brain damage through “bystander suppression.”

Administration

One aspect of the current invention is a method for inducing E-selectintolerance in a subject. This method involved administering E-selectin tomucosal tissues of a subject. According to the invention any E-selectinthat can induce immune tolerance in the subject to E-selectin can beused. Thus, for example, an E-selectin with any of SEQ ID NO: 1-26,30-33 can be used in the methods and compositions of the invention.

Tolerance to an antigen such as E-selectin can be induced byadministration to many types of mucosal tissues including oral, nasal,enteral, vaginal, rectal and respiratory mucosa. By reducing enzymaticdegradation in the gastrointestinal tract, lower doses of antigen maysometimes be used for non-oral routes of administration. In someembodiments, tolerance is induced by intranasal administration ofE-selectin.

Tolerance, including bystander tolerance, can be induced by a singleseries of E-selectin administrations. Thus, for example, E-selectintolerance or E-selectin bystander tolerance can be induced by anadministration protocol involving a single series of fiveadministrations of E-selectin over a period of two weeks. In otherembodiments, this regimen of five administrations over two weeks isrepeated at least once. Repeating a series of E-selectin administrationis referred to herein a “booster” series of administrations. Thus, asingle series of E-selectin dosages is administered within about one totwo weeks. The “booster” administrations repeat this series ofE-selectin administrations after a period of several weeks without anyE-selectin administrations. In some embodiments, this booster regimen isrepeated every three weeks for the remainder of the life of the subject.

Dosages, E-selectin sources, formulations, dosage volumes, regimens, andmethods for analyzing results aimed at optimizing E-selectin tolerancecan vary. Thus, minimum and maximum effective dosages vary depending onthe method of administration. Suppression of the clinical andhistological changes associated with vascular dementia can occur withina specific dosage range, which, however, varies depending on theorganism receiving the dosage, the route of administration, whetherE-selectin is administered in conjunction with other co-stimulatorymolecules, and the specific regimen of E-selectin administration. Forexample, in general, nasal administration requires a smaller dosage thanoral, enteral, rectal, or vaginal administration.

When E-selectin is administered mucosally, dosages are used that rangefrom about 0.005 to about 500 mg/day, or from about 0.05 to about 50mg/day. In some embodiments, mucosal dosages are from about 0.5 μg toabout 50 mg per administration, or from about 0.5 μg to about 5 mg peradministration. In view of the guidelines provided herein, optimizationof the dosage necessary for immune suppression involves no more thanroutine experimentation.

E-selectin formulations of the present invention may comprise inertconstituents including pharmaceutically-acceptable carriers, diluents,solubilizing agents, emulsifying agents, salts, and the like, as areavailable in the art. Preferred E-selectin formulations are intranasalformulations including normal saline solutions, such as, for example,isotonic and physiologically buffered saline solutions andphosphate-buffered saline (PBS) solutions. The total volume of theintranasal formulations is typically less than 1 milliliter, preferablyless than 100 μl.

For oral or enteral E-selectin formulations for use with the presentinvention, tablets may be formulated in accordance with conventionalprocedures employing solid carriers well-known in the art. Capsulesemployed for oral formulations to be used with the methods of thepresent invention may be made from any pharmaceutically acceptablematerial, such as gelatin or cellulose derivatives. Sustained releaseoral delivery systems and/or enteric coatings for orally administereddosage forms are also contemplated, such as those described in U.S. Pat.No. 4,704,295, “Enteric Film-Coating Compositions,” issued Nov. 3, 1987;U.S. Pat. No. 4,556,552, “Enteric Film-Coating Compositions,” issuedDec. 3, 1985; U.S. Pat. No. 4,309,404, “Sustained Release PharmaceuticalCompositions,” issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406,“Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982.

Examples of solid carriers include starch, sugar, bentonite, silica, andother commonly used carriers. Further non-limiting examples of carriersand diluents which may be used in the formulations of the presentinvention include saline, syrup, dextrose, and water.

E-selectin can also be administered in an aerosol or inhaled form.Examples of formulations for tolerizing agents administered byinhalation are provided in Weiner, H. et al., “Improved treatment ofautoimmune diseases by aerosol administration of auto antigens,”WO9108760 (1991). The antigens can be administered as dry powderparticles or as an atomized aqueous solution suspended in a carrier gas(e.g., air, N.sub.2, and the like).

Dry aerosol in the form of finely divided solid particles of E-selectinthat are not dissolved or suspended in a liquid can also be used in thepractice of the present invention. E-selectin formulations may be in theform of dusting powders and comprise finely divided particles having anaverage particle size of between about 1 and 5 microns, preferablybetween 2 and 3 microns. Finely divided particles may be prepared bypulverization and screen filtration using techniques available in theart. The particles may be administered by inhaling a predeterminedquantity of the finely divided or powdered material.

The E-selectin formulations of the present invention may also beadministered in soluble form as an aerosol spray using, for example, anebulizer such as those described in U.S. Pat. No. 4,624,251 issued Nov.25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No.3,561,444 issued Feb. 9, 1971; and U.S. Pat. No. 4,635,627 issued Jan.13, 1971. Other systems of aerosol delivery, such as the pressurizedmetered dose inhaler (MDI) and the dry powder inhaler (see, e.g.,Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds.pp. 197-224, Butterworths, London, England, 1984) can be used whenpracticing the present invention.

One useful animal model for the analysis of E-selectin formulations andtheir effectiveness in treating or preventing stroke is the stroke-proneand spontaneously hypertensive SHR-SP rat (Okamoto, K. et al.,“Establishment of the stroke-prone spontaneously hypertensive rat(SHR),” Circ. Res. (Suppl.) 34, 35: 1 (1974)). SHR-SP rats may beobtained from professor Yukio Yamori, Graduate School of Human andEnvironmental Studies, Kyoto University, Yoshida Nihonmatsu-cho,Sakyo-ku, Kyoto, 606-8316, Japan. SHR-SP rats typically die ofearly-onset cardiovascular disease, sometimes as early as 14 weeks ofage, although some SHR-SP rats live to more than 56 weeks of age.Frequently, the cardiovascular disease manifests as a stroke in theserats. The occurrence of a stroke in these rats is diagnosed by measuringbehavioral status that could be divided into 4 patterns: noabnormalities (grade 1), irritable (grade 2), lethargic (grade 3),akinetic (grade 4) (Yamori, U. et al., Japanese Circulation Journal 46:274 (1982)).

The brains of SHR-SP rats at the time of death, typically containnumerous infarcts and intraparenchymal hemorrhage areas that can becounted and measured through microscopic analysis of brain sections.Thus, the effectiveness of an E-selectin formulation can be determinedby comparing infarct and intraparenchymal hemorrhage numbers and areasin SHR-SP rats that have been treated with control or test E-selectinformulations. The administration regimen can be evaluated in a similarmanner. The control formulations can consist of only carrier componentsor non-specific antigens (e.g., ovalbumin). In addition, the efficacy ofa regimen of booster administrations versus a single series ofE-selectin administration can be compared. Examples of these proceduresand comparisons are disclosed in the Examples section of thisspecification.

Another useful animal model for the analysis of E-selectin formulationsand their effectiveness in treating or preventing vascular dementiainvolves occlusion of carotid arteries in rats. See, e.g., Sarti et al.,Persistent impairment of gait performances and working memory afterbilateral common carotid artery occlusion in the adult Wistar rat,BEHAVIOURAL BRAIN RESEARCH 136: 13-20 (2002). Thus, cerebrovascularwhite matter lesions can be experimentally induced in the rat brain as aresult of chronic cerebral hypoperfusion. This model is created bypermanent occlusion of both common carotid arteries. For example, Wistarrats can be anesthetized, the bilateral common carotid arteries areexposed through a midline cervical incision and the common carotidarteries are double-ligated with silk sutures bilaterally. The cerebralblood flow (CBF) then initially decreases by about 30 to 50% of thecontrol after ligation. The CBF values later range from 40 to 80% ofcontrol after about 1 week to about 1 month.

As illustrated herein, white matter rarefaction is detected in rats withoccluded carotid arteries after 3 days or more of ligation Microglia andastroglia were activated after arterial occlusion in a manner thatpredicts the extent and severity of the subsequent white matter damages.A few lymphocytes, labeled with CD4 or CD8 antibodies, can be detectedas scattered in the white matter after arterial occlusion. Otherpathological changes include axonal damage and demyelination in whitematter lesions. Blood-brain barrier disruptions have also been observedas well as increased matrix metalloproteinase activity in white matterlesions. These changes appear very similar to those in humancerebrovascular white matter lesions. Moreover, these results suggestthat inflammatory and immunologic reactions play a role in thepathogenesis of the white matter changes.

Such physiological changes are correlated with learning and memoryproblems in the occluded carotid artery rat model. Thus, the gaitperformance of rats with occluded arteries declines over time incomparison with baseline. At 60 and 90 days, rats with bilateral commoncarotid artery occlusion showed decreased performances on objectrecognition and Y maze spontaneous alternation test in comparison withsham-operated rats. Thus, this rat model of experimental chroniccerebral hypoperfusion by permanent occlusion of the bilateral commoncarotid arteries exhibited significant learning impairments along withrarefaction of the white matter. This model is a useful tool to assessthe effectiveness of E-selectin tolerization on the pathophysiology ofchronic cerebral hypoperfusion, and to provide data for determiningoptimal dosages and dosage regimens for preventing the cognitiveimpairment and white matter lesions in patients with cerebrovasculardisease.

The effectiveness of an E-selectin formulation for treating orpreventing vascular dementia can therefore be determined by observingthe gait performance, memory, learning abilities and the incidence andseverity of white matter lesions in rats with carotid artery occlusions.Similarly, the E-selectin dosage and administration schedule can beadjusted pursuant to the memory and learning abilities of human patientsbeing treated for vascular dementia.

Assessment of the effect of E-selectin formulations on an immuneresponse to E-selectin can also be made, for example, by determiningdiminution in certain inflammation markers, such as the number ofactivated T-cell clones directed against activated vascular tissue.Immunological tolerance can be measured by a number of methods that arewell-known in the art. In one preferred embodiment, delayed typehypersensitivity (DTH) response can measured in animals by injectingE-selectin, for example, into the footpad or ear flap of an organism tobe analyzed and then administering a challenge injection, for exampleinto a footpad or an ear, at a later time, typically more than 1 weeklater, most preferably 2 weeks later. DTH reactions can be measuredafter the elicitation injection as the increase in swelling at the siteof the antigen re-challenge. Footpad or ear swelling can be measured at,for example, 0, 24 and 48 hr after challenge.

In some embodiments, the optimum dosage of E-selectin is one thatinduces E-selectin tolerance, for example, bystander tolerance. In otherembodiments, the optimum dosage of E-selectin is one that generates themaximum protective effect in preventing vascular dementia, stroke andthe like. In other embodiments, the optimum dosage of E-selectin is onegenerating the maximum beneficial effect on damaged tissue caused byarterial occlusion. An effective dosage causes at least a statisticallyor clinically significant attenuation of at least one marker, symptom,or histological evidence characteristic of vascular dementia. Markers,symptoms and histological evidence characteristic of vascular dementiainclude memory loss, confusion, disturbances in axonal transport,demyelination, induction of metalloproteinases (MMPs), activation ofglial cells, infiltration of lymphocytes, edema, inflammation andimmunological reactions that lead to tissue damage and further vascularinjury. Stabilization of symptoms or diminution of tissue damage, underconditions wherein control patients or animals experience a worsening ofsymptoms or tissue damage, is one indicator of efficacy of a suppressivetreatment.

Ascertaining the effective dosage range as well as the optimum amount ofE-selectin is accomplished using the teachings of the presentapplication as well as any available teachings in the art. For example,an optimum regimen for administering E-selectin is determined in lightof the information disclosed herein and well known informationconcerning administration of bystander antigens and autoantigens.Routine variation of dosages, combinations, and duration of treatment isperformed under circumstances wherein the effects of such variations onthe organism can be measured.

For example, dosages for mammals and humans can be determined bybeginning with a relatively low dose (e.g., 1 microgram) andprogressively increasing the dosage while measuring appropriateresponses (e.g., number of TGF-β, IL-4, and/or IL-10 secreting cells;number and activation of immune attack T-cells in the blood (e.g., bylimiting dilution analysis and ability to proliferate); and/or diseaseseverity). The optimum dosage provides maximal prevention from vasculardementia or the maximum protection from tissue damage caused by vascularocclusion while minimizing undesirable side effects. Potential sideeffects include the generation of pathogenic autoantibodies (Hu, W. etal., “Experimental mucosal induction of uveitis with the 60-kDa heatshock protein-derived peptide 336-351,” Eur. J. Immunol. 28: 2444(1998); Genain C. P., et al., “Late complications of immune deviationtherapy in a nonhuman primate,” Science 274: 2054 (1996)) or a cytotoxicT lymphocyte response that induces autoimmunity (Blanas E., et al.,“Induction of autoimmune diabetes by oral administration ofautoantigen,” Science 274: 1707 (1996)).

An effective dosage causes at least a statistically or clinicallysignificant attenuation of at least one symptom of vascular dementia, orat least a statistically or clinically significant attenuation of theoccurrence rate or time to onset of vascular occlusion. The maximumeffective dosage of a bystander antigen in humans can be ascertained bytesting progressively higher dosages in clinical trials starting with arelatively low dosage, for example 0.5 μg per administration.

Preferred dosages for intranasal instillations are from about 0.5 toabout 50 mg per administration, preferably for humans approximately fromabout 0.5 μg to 5 mg per administration. For rats, one preferred dosageis 5 μg per administration. Preferred aerosol pharmaceuticalformulations may comprise, for example, a physiologically-acceptablebuffered saline solution containing between about 0.1 mg and about 300mg, or about 1 mg and about 300 mg of E-selectin.

In some embodiments, E-selectin is administered in a series ofadministrations. Typically these administrations are spaced apart over aperiod of 1 to 2 weeks. For example and as further detailed in theExamples, E-selectin can be administered in five intranasaladministrations over a two week period. This protocol can involveadministering E-selectin every other day for ten days. Preferably, theadministration regimen is repeated in booster administrations that aregenerally administered several weeks apart. In one embodiment, boosteradministrations are given after every three weeks. Boosteradministrations may include a series of administrations, as describedabove for initial administrations.

Cytokine and non-cytokine synergists can be used in conjunction withE-selectin in the present invention to enhance the effectiveness ofE-selectin tolerization. Administration “in conjunction with”encompasses simultaneous and sequential administration, as well asadministration in combined form or separately. Oral and parenteral useof cytokine synergists (Type I interferons) has been described inHafler, D. A. et al., “Treatment of autoimmune disease using oraltolerization and/or type 1 interferon,” WO9527499 (1995). Administrationof Th2 enhancing cytokines is described in Weiner H. L., et al.,“Treatment of autoimmune disease using oral tolerization and/orTh2-enhancing cytokines,” WO95275000 (1995). For example, IL-4 and IL-10can be administered in the manner described in Weiner et al. Id.

Non-limiting examples of non-cytokine synergists for use in the presentinvention include bacterial lipopolysaccharides from a wide variety ofgram negative bacteria such as various subtypes of E. coli andSalmonella (LPS, Sigma Chemical Co., St. Louis, Mo.; Difco, Detroit,Mich.; BIOMOL Res. Labs., Plymouth, Pa.), Lipid A (Sigma Chemical Co.,St. Louis, Mo.; ICN Biochemicals, Cleveland, Ohio; Polysciences, Inc.,Warrington, Pa.); immunoregulatory lipoproteins, such as peptidescovalently linked to tripalmitoyl-5-glycarylcysteinyl-seryl-serine(P.sub.3 C55) which can be obtained as disclosed in Deres, K. et al.(Nature, 342: 561-564, “In vivo priming of virus-specific cytotoxic Tlymphocytes with synthetic lipopeptide vaccine,” 1989) or “Braun's”lipoprotein from E. coli which can be obtained as disclosed in Braun,V., Biochim. Biophys. Acta 435: 335-337, 1976; and choleratoxin.beta.-chain (CTB) the synergist ability of which has beendescribed (though not in connection with abatement of autoimmunereaction) by Sun, J-B et al., “Cholera toxin B subunit: an efficienttransmucosal carrier-delivery system for induction of peripheralimmunological tolerance,” Proc. Natl. Acad. Sci. (USA) 91: 10795 (1994).The effective dosage range for non-cytokine synergists for mammals isfrom about 15 ng to about 15 mg per kg weight and preferably 300 ng-12mg per kg weight. The effective dosage range for oral Type I interferonfor mammals is from 1,000-150,000 units with no maximum effective dosagehaving been discerned. Another active compound that may be useful incombination with E-selectin is methotrexate which is known to cause amarked Th2 immune deviation with greatly increased IL-4 secretion whengiven on a pulse regimen (Weiner et al., “Treatment of AutoimmuneDisease Using Tolerization in Combination with Methotrexate,” U.S. Pat.No. 5,935,577 (1999).

Ascertaining the optimum regimen for administering E-selectin and/or theco-stimulatory molecule is determined in light of the informationdisclosed herein and well known information concerning administration ofbystander antigens and autoantigens. Routine variation of dosages,combinations, and duration of treatment is performed under circumstanceswherein the effects of such variations on the organism can be measured.The co-stimulatory agent is preferably administered within 24 hours ofadministration of E-selectin. More preferably, it is administered at thesame time as E-selectin. Most preferably, both are administered in acombined oral formulation.

The following examples describe and illustrate the methods andcompositions of the invention. These examples are intended to be merelyillustrative of the present invention, and not limiting thereof ineither scope or spirit. Those of skill in the art will readilyunderstand that variations of the materials used in, and the conditionsand processes of, the procedures described in these examples can beused.

Example 1 Reduction of Brain Infarcts by Administration of E-Selectin

This Example illustrates the effects of administering E-selectin onreducing the incidence and size of infarcts in the brains ofstroke-prone rats. Further information on stroke treatment by E-selectintolerization can be obtained in a related application, PCT ApplicationSer. No. PCT/US01/16583, which is incorporated by reference herein inits entirety.

Materials and Methods

Male and female stroke-prone and spontaneously hypertensive (SHR-SP)8-10 week-old rats were obtained from the NIH colony. Okamoto (1974)Circ. Res. (Suppl.) 34, 35: 1 (1974). At 11 weeks of age, soluble humanE-selectin (encoding the following domains: human E-selectin lectin,EGF, CR1, CR2 with a myc peptide tail), ovalbumin or vehicle (PBS) wereadministered intranasally. Purified human E-selectin was obtained fromProtein Design Laboratories (Fremont, Calif.).

E-selectin and control preparations were administered in the followingmanner: SHR-SP rats were divided into three groups: (1) a saline (PBS)control group, (2) an E-selectin administration group (ES group), and(3) an ovalbumin (OVA) administration group (OVA group). In addition, ESand OVA groups were divided into single (non-booster) and repetitive(booster) administration groups. For the control group, 20 μl ofphosphate-buffered saline (PBS) was administered into each nostril everyother day for 10 days for a total of 5 administrations. For the ESnon-booster group, 2.5 μg E-selectin in 20 μl PBS was administered intoeach nostril every other day for 10 days for a total of 5administrations. For the ES booster group, an initial 2.5 μg ofE-selectin in 20 PBS was administered as above for the non-boostergroup; additionally, 2.5 μg of E-selectin in 20 μl of PBS wasadministered intranasally into each nostril every other day for 10 days(3 weeks after the first E-selectin course) and repeated every 3 weeksuntil the animals were sacrificed. For the OVA non-booster group, 2.5 μgovalbumin in 20 PBS was administered into each nostril every other dayfor 10 days for a total of 5 administrations. For the OVA booster group,an initial 2.5 μg of ovalbumin in 20 μl PBS was administered into eachnostril as above for the non-booster group; additionally, 2.5 μg ofovalbumin in 20 μl of PBS was administered intranasally into eachnostril every other day for 10 days (3 weeks after the first ovalbumincourse) and repeated every 3 weeks until the animals were sacrificed.

The rats were evaluated for physical and neurological signs of stroke.These evaluations included an assessment of excitement (i.e.,piloerection, hyperkinesis), hyperirritability (i.e., jumping, trying toescape), behavioral and psychological depression (i.e., hypokinesis,hyposthenia, hyporesponsiveness), motion disturbance (i.e., transientepisode of repetitive lifting of paws, ataxia, paresis, paralysis), andlate symptoms observed near the time of death (i.e., apathy, coma,urinary incontinence). The rats were also monitored by measuringarterial blood pressure, body weight, heart weight, and arterial bloodgas using methods available in the art.

Infarcts were evaluated in the following manner. When animals showedsigns of cardiac failure, kidney failure, or stroke, they were perfusedand their brains were removed for histology and image processing.Sections from 8 predetermined stereotactic levels were cut from eachbrain (total of 240 sections). The number and area of infarcts orhemorrhages were determined for each section from each animal.Statistical significance of E-selectin administrations was determined bycomparing E-selectin groups to control groups by a Cox ProportionalHazards Model.

The animals lived for variable periods from 14 weeks to the terminationof the experiment at 56 weeks. Deaths were caused by heart failure andkidney failure secondary to severe hypertension (mean systolic bloodpressure 215 mm Hg), as well as by strokes. Average age at time of deathand average systolic blood pressure did not differ among theexperimental groups.

Results

The experimental group of animals that received E-selectin with boosteradministrations had a statistically significant reduction in thefrequency and area of infarcts compared to control groups (p<0.0001).Mean area of infarcts decreased from between about 6.873 mm² to about27.718 mm² in control and single administration E-selectin groups toabout 0.002 mm² in the E-selectin booster group (i.e., a greater than99% reduction; see Tables I-IV). Mean number of infarcts decreased fromabout 3.0 to about 7.3 for control and single administration E-selectingroups to about 0.3 in E-selectin booster groups (i.e., a greater than91% reduction; see Tables I-IV). Intraparenchymal hemorrhages wereabsent from the E-selectin booster group, but were present at an averagenumber of from about 3.2 to about 2.3 per brain section analyzed incontrol and single E-selectin administration groups (see Tables I-IV).

TABLE I Group OVA Data Sample Infracts Intraparenchymal Hemorrhage (sex)Number Area (mm²) Number Area (mm²) 1 (female) 13 6.966 2 0.439 2(female) 0 0 0 0 3 (female) 1 0.062 15 0.390 4 (female) 19 133.850 40.950 5 (female) 15 70.559 1 0.02{grave over ( )} 6 (female) 10 10.308 00 7 (female) 0 0 0 0 8 (female) 0 0 0 0 mean 7.3 27.718 2.8 2.25

TABLE II Group OVAb Data Sample Infracts Intraparenchymal Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (female) 0 0 0 0 2 (female)3 0.734 1 4.784 3 (female) 21 40.502 17 1.372 4 (female) 0 0 0 0 5(female) 0 0 1 0.063 6 (female) 0 0 0 0 mean 4.0 6.873 3.2 1.037

TABLE III Group ES Data Sample Infracts Intraparenchymal Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (female) 0 0 0 0 2 (male) 00 0 0 3 (female) 9 13.488 5 0.177 4 (female) 14 77.909 13 7.553 5(female) 0 0 0 0 6 (female) 1 0.012 0 0 7 (male) 0 0 0 0 8 (male) 0 0 00 mean 3.0 11.426 2.3 0.966

TABLE IV Group ESb Data Sample Infracts Intraparenchymal Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (male) 0 0 0 0 2 (female) 00 0 0 3 (female) 0 0 0 0 4 (female) 0 0 0 0 5 (male) 1 0.003 0 0 6(female) 0 0 0 0 7 (female) 1 0.011 0 0 8 (male) 0 0 0 0 mean 0.3 0.0020 0

Example 2 Induction of Tolerance to E-Selectin

This Example provides data showing that tolerance to E-selectin wasinduced by the intranasal administration protocol of E-selectindescribed above, which resulted in decreased stroke-related tissuedamage.

For this analysis, either E-selectin or control PBS preparations wereadministered to rats as described in Example 1 for the non-boostergroups. Thus, 2.5 μg E-selectin in 20 μl PBS was administered into eachnostril every other day for 10 days for a total of 5 administrations.

Fourteen days after intranasal administration to induce tolerization,delayed-type hypersensitivity (DTH) was analyzed by injecting 5 μg ofE-selectin in 50 μl of PBS and 50 μl of complete Freund's adjuvant intohindpads (s.q.). Another fourteen days later, the rats werere-challenged by injecting 5 μg E-selectin in 50 μl PBS into the ear.Ear thickness was measured with microcalipers (Mitsutoyo) 48 hours laterto assess to degree of tolerization to E-selectin.

Results of the delayed-type hypersensitivity assay demonstrated thatintranasal instillation of human E-selectin induced tolerance.Administration of E-selectin intranasally before footpad and earinjection resulted in a significant suppression of ear swelling comparedto control groups, as measured with Mitsutoyo microcalipers. Inparticular, rats “tolerized” with PBS exhibited a an approximate 55%change in ear thickness (about 0.36 mm swelling), while the E-selectintolerized rats exhibited only about a 20% change in ear thickness (about0.11 mm swelling). The difference was statistically significant at thep<0.01 level.

These data demonstrate that the E-selectin administration protocol usedinduced tolerance to E-selectin.

Example 3 Vascular Dementia Animal Model

This Example provides information about the animal model used forevaluation of vascular dementia and the effects of E-selectintolerization on vascular dementia.

The experimental model used for vascular dementia was hypoperfusion ofWistar rat brains. In particular, previous work has shown thatcerebrovascular white matter lesions can be experimentally induced inthe rat brain as a result of chronic cerebral hypoperfusion and thatsuch hypoperfusion leads to impaired memory. See Sarti et al.,Persistent impairment of gait performances and working memory afterbilateral common carotid artery occlusion in the adult Wistar rat,BEHAVIORAL BRAIN RESEARCH 136: 13-20 (2002). This model is created bypermanent occlusion of both common carotid arteries as described below.

The animals were anesthetized with 5% isoflurane for induction and 1.5%isoflurane for maintenance in 30% O₂/70% N₂O by facemask. The core bodytemperature was monitored and maintained at 37.0±0.5° C. using a heatingpad and a heating lamp. Through a midline cervical incision, both commoncarotid arteries were exposed and double-ligated with 5-0 silk suturesas previously described by Wakita H., Acta Neuropathol. (Berl) 1994; 87:484-492. After the operation, the rats were kept in cages with food andwater ad libitum. As controls, four animals were subjected to the samesurgical procedures without bilateral carotid ligation.

Cerebral blood flow (CBF) after carotid artery occlusion was 30 to 50%of the control several days after ligation. The CBF decreased to valuesranging from 40 to 80% of control over a prolonged period (1 week-1month).

The effects of carotid artery occlusion upon brain tissues areillustrated in FIGS. 6-13 by comparing results for the PBS treatedanimals, who received bilateral carotid ligation after administration ofPBS during the tolerization schedule (shown in FIG. 1), with thesham-operated animals that did not receive bilateral carotid ligation.As shown FIGS. 6-8, white matter becomes rarefied after carotid arteryocclusion (compare PBS vs. Sham-operated tissue sections in FIG. 6-7 andgraphic summary in FIG. 8).

FIG. 9 shows that glial cells become activated in white matter aftercarotid artery occlusion. In particular, as summarized in FIG. 10,significantly (p=0.177) greater numbers of MHC class II immunopositivemicroglia are observed in rats who received bilateral carotid ligation(“PBS” rats) than in rats that did not receive bilateral carotidligation (“Sham” operated rates). Thus, microglia and astroglia wereactivated briefly after vascular occlusion. Moreover, such activationwas predictive of the extent and severity of the subsequent white matterdamage.

Additional lymphocytes were detected with CD4 or CD8 antibodies (FIG. 4)after occlusion of carotid arteries. As shown in FIG. 11, greaternumbers of CD4 positive T cells infiltrated the corpus callosum of ratswho received bilateral carotid ligation (“Ligated” rats) than wasobserved in rats that did not receive bilateral carotid ligation (“Sham”operated rats). These CD4 or CD8 positive T cells were scattered in thewhite matter after occlusion. These changes are similar to those inhuman cerebrovascular white matter lesions and suggest that inflammatoryand immunologic reactions play a role in the pathogenesis of the whitematter changes.

FIG. 12 show that the numbers of TNF-α immunopositive blood vesselsincrease after carotid artery occlusion. In particular, as summarized inFIG. 13, significantly (p=0.002) greater numbers of TNF-α immunopositiveblood vessels are observed in rats who received bilateral carotidligation (“PBS” rats) than in rats that did not receive bilateralcarotid ligation (“Sham” operated rates).

These data indicate that immunological activity accompanies brain damageafter carotid artery occlusion. The effects of carotid artery occlusion(ischemia) upon brain function are summarized in FIG. 16.

Example 4 E-Selectin Administration Ameliorates Vascular Dementia

This Example illustrates that mucosal tolerization to E-selectinprotects against several forms of memory dysfunction and white matterdamage in the rat model of vascular cognitive impairment.

Materials and Methods

Animals: A total of 34 Male and female Wistar rats (Charles RiverLaboratories, Wilmington, Mass., USA) aged 9 weeks were used. TheNational Institute of Neurological Disorders and Stroke Animal Care andUse Committee approved all experiments.

Tolerization Schedule: Animals were divided into two groups. Intranasalapplication of E-selectin was carried out with the animals under briefanesthesia with 5% isoflurane in 30% O₂/70% N₂O. For some experimentsE-selectin with SEQ ID NO: 30 or SEQ ID NO: 31 was used. However, thesesequences contain a histidine tag sequence (GGASTRAAEQKLISEEDLNGTRSGHHHHHH (SEQ ID NO: 29)), which is used for manufacturingpurposes. E-selectin without the histidine tag (e.g., E-selectin withSEQ ID NO: 8 or 32, or any of SEQ ID NO: 5-8, 18, 19, 30-33) may be moredesirable for therapeutic purposes.

Intranasal instillations to animals in groups 1 and 2 were as follows:

(1) Control rats received PBS (Quality biological, Inc, GaithersburgMd., USA)

(2) Experimental rats received recombinant human E-selectin (Novavax,Rockville Md., USA)

The tolerization schedule involved a single series of administrations ora single series of administrations plus a booster series ofadministrations as follows (see FIG. 1):

(1) Single or non-booster administration schedule: PBS (20 μl) orE-selectin (2.5 μg/20 μl) was instilled into each nostril every otherday for 10 days (total of 5 administrations) (FIG. 1).

(2) Booster administration schedule: intranasal instillations of thesame substance at the same volume and concentration on the same scheduleas described for the single or non-booster schedule described above, butthe administrations were repeated at 3-week intervals from 1 monthbefore surgery to 3 months after surgery (FIG. 1).

Delayed-Type Hypersensitivity Reaction: For assessing the delayed—typehypersensitivity reaction, a single-course tolerization schedule witheither PBS or E-selectin was conducted (n=4) (as shown in FIG. 1 for thenon-booster group). Fourteen days later, the animals were immunized(hind footpad) with 75 μg E-selectin/200 PBS plus 50 μl completeFreund's adjuvant (Sigma, St. Louis, Mo.). Fourteen days after that, earthickness was measured and the animals were re-challenged with 75 μgE-selectin/100 μl PBS injected into the ear. Ear thickness increase overbaseline was measured with microcalipers (Mitsutoyo Co, Ltd, Kawasaki,Kanagawa, Japan) 2 days later.

Surgery: The booster tolerization schedule was repeated at 3-weekintervals from 1 month before the surgery to 3 months after the surgery.In order to adjust the surgery workload, half of the rats from eachgroup were randomly selected and subjected to the surgery 3 days afterthe last dose of the first booster tolerization schedule; and surgerywas performed 4 days after the last dose of the first boostertolerization schedule for the remaining half of rats.

The animals were anesthetized with 5 isoflurane for induction and 1.5%isoflurane for maintenance in 30% O₂/70% N₂O by facemask. The core bodytemperature was monitored and maintained at 37.0±0.5° C. using a heatingpad and a heating lamp. Through a midline cervical incision, both commoncarotid arteries were exposed and double-ligated with 5-0 silk suturesas previously described by Wakita H., Acta Neuropathol. (Berl) 1994; 87:484-492. After the operation, the rats were kept in cages with food andwater ad libitum. As controls, four animals were subjected to the samesurgical procedures without bilateral carotid ligation.

Behavioral assessment: Behavioral assessment consisted of objectrecognition, T-maze spontaneous alternation, and T-maze left/rightdiscrimination memory retention tests. An observer who was blind to thetreatments performed behavioral assessment.

Object Recognition test: This test evaluates non-spatial working memoryrelated to the frontal subcortical circuits (Ennaceur A. Behav Brain Res1988; 31:47-59, Sarti C, Behav Brain Res. 2002; 136:13-20). Theapparatus was formed by a glass box (30×60×30 cm). The apparatus wasilluminated by a 100 W lamp suspended 70 cm above the box in a darkenedroom. The day before testing, rats were habituated to the testenvironment by exploring the box for 6 min without objects. On the dayof the test, a session of two trials was given. The inter-trial intervalwas 60 min.

In the first trial, two identical objects were placed on the centerlineof the long axis of the floor, 5 cm from each end of the apparatus. Ratswere placed into the center of the box and allowed to explore the twoobjects for 6 min. The amount of time spent exploring each object wasrecorded. During the second trial, one of the objects presented in thefirst trial is replaced by a novel object and rats are left in the boxfor 6 min. The time spent for the exploration of the familiar (Tf) andthe novel object (Tn) is recorded separately. Exploration is consideredsniffing at the object within a distance of 2 cm from the object and/ortouching it with the nose. A discrimination index (Tn-Tf/Tn+Tf) iscalculated.

For each animal, one pair of objects in the first trial was selected atrandom from a set of three plastic objects that differed in shape andcolor (red cubes, green pyramids, and blue cylinders of 6 cm height),and the role (familiar and novel object) and the position of the twoobjects in the second trial were randomly changed to avoid object andplace preference. After each exposure, the apparatus and the objectswere cleaned carefully with 70% alcohol to avoid olfactory stimuli.

T-maze spontaneous alternation: This test evaluates spatial workingmemory related to the frontal subcortical circuits (Bartolini L.,Pharmacol Biochem Behay. 1992; 43:1161-1164, Sarti C, Behav Brain Res.2002; 136:13-20). Spontaneous alternation was investigated in an acrylicT shaped runway. It consisted of a start box (20×18 cm) and start arm(60 cm long), and two identical goal arms (both 50 cm long). All armswere 10 cm wide and 10 cm high. Spontaneous alternation refers to theinstinctive behavioral tendency by which rats typically alternate theirchoices between the arms of the T-maze more often than they repeat theirinitial choice. Rats were placed in the start box of the T-maze and amaximum time of 5 min was allowed for them to explore the maze.Spontaneous alternation was defined as following: the rat entered withall four feet into one goal arm, came back, and then entered with allfour feet into the opposite goal arm. The number of rats who alternatedwas recorded.

T-maze left/right discrimination memory retention: This test evaluatesspacial reference memory related to the hippocampus and caudoputamen(Oliveira M G, Neurobiol Learn Mem 1997; 68:32-41). This test wasrepeated at 2, 6 and 10 weeks after surgery. The dimensions of theT-maze apparatus were described above. The exit of the start box and theentrances of the goal arms could be blocked by guillotine doors. Carefulconsideration was given to avoid providing the animals with any spatialcues. To minimize olfactory cues, the maze was wiped carefully aftereach run with 70% alcohol.

Training sessions for left/right discrimination memory retention: Theday before training, after the spontaneous alternation test, rats werehabituated for 15 min to the presence of food pellets (Bacon Softies;Bio-Serv, Frenchtown, N.J., USA) placed at the end of each arm in theT-maze. On days 1 to 3, the rats were food-deprived for 8 to 12 hourseach day before the T-maze left/right discrimination training. Thistraining consisted of 3 stages. In the performance of the training, halfof the rats from each group were randomly selected and reinforcement(food reward) placed on the right arm; for the other half of the ratsfrom each group, the reinforcement was placed on the left arm. Thereinforced arm then remained consistent throughout the training period.The first stage consisted of 5 trials. In this stage, a guillotine doorwas placed to close off one arm, and the animal was forced to enter theopen arm, which was baited with a food reward that the animal wasallowed to eat. For all runs the animals remained on the maze until 2min had elapsed; they were then placed in the start box for 2 min. Thesecond stage consisted of 5 trials. In this stage, a guillotine door wasplaced to close off the same arm as that in the first stage, and theanimal was forced to enter the open arm, which was not baited with afood reward. When the animal entered into the open arm, a food rewardwas given and the animal was allowed to eat the food. The animalsremained on the maze for 2 min and were then placed in the start box for2 min. In the third stage, a guillotine door was removed, and the animalcould enter into either arm (correct side and incorrect side). If theanimal chose the arm on the correct side the animal received a foodreward and was allowed to eat for 2 min after which it was placed in thestart box for 2 min. If the animal chose the incorrect side-arm, theanimal was picked up immediately and placed in the start box for 2 min.The third stage was continued until the animals made 4 consecutivecorrect choices or until they had had 20 training sessions (the trainingceiling). This procedure was performed daily on three successive days(on days 1 to 3).

Left/right discrimination memory retention test session: The retentionof left/right discrimination memory was evaluated at 1, 2, 3, 5, 7, 10and 14 days after the training session. The animals were given 10 trialson each testing day. An entry was defined as all four paws entering thearm. The total number of correct entries was recorded.

Histopathology: At 90 days after surgery, the animals were deeplyanesthetized with sodium pentobarbital (100 mg/kg, intraperitoneally),perfused transcardially with 0.01 M PBS, and then perfused with afixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB,pH 7.4). Coronal brain blocks including the caudoputamen or optic nervewere embedded in paraffin for histological examination. Twomicrometer-thick paraffin sections were then cut on a microtome. Theluxol fast blue stain was used to evaluate the myelin damage.Immunocytochemistry with the cocktail of monoclonal antibodies directedagainst non-phosphorylated neurofilaments (SMI 311, Covance ResearchProducts, Inc., Berkeley, Calif., USA) was used for the assessment ofaxonal injury (Rosenfeld J, J Neuropathol Exp Neurol. 46:269-282(1987)). The sections were incubated for 1 hr in 0.1 M PBS containing0.3% Triton X-100 for permeabilization. Ten percent donkey serum wasapplied for blocking followed by incubation overnight in primaryantibody (SMI 311) in a dilution of 1:500. The sections weresubsequently incubated with a biotinylated anti-mouse IgG raised indonkey (Jackson Immuno Research Labs, West Grove, Pa., USA, 1:2000) for1 hr, and then incubated with an avidin-biotin peroxidase complexsolution (Vector Laboratories, Burlingame, Calif., USA, 1:100) for 1 hr.After each incubation, the sections were rinsed for 30 min with 0.1 MPBS containing 0.3% Triton X-100. The immunoreaction products werevisualized with diaminobenzidine (DAB kit, Vector Laboratories,Burlingame, Calif., USA). The severity of the white matter lesions wasevaluated by the fiber density of luxol fast blue-stained sections.Monochromatic photo images of both sides of the corpus callosum, thetraversing fiber bundles of the caudoputamen bilaterally and both opticnerves were taken by means of a microscope with a x40 objectiveconnected to a digital camera (MetaMorph Image Processing System,Universal Imaging Corp, Downingtown, Pa., USA). These images wereconverted into PICT files by Photoshop (Adobe Systems Incorporated, SanJose, Calif., USA) and the fiber density of each PICT file was analyzedwith the NIH image computer program. To account for the variation of thefiber density between right and left sides of the corpus callosum andcaudoputamen, the average of the fiber densities of both sides wascalculated.

For free-floating immunohistochemistry, the rest of the coronal blockswere post-fixed for 12 hrs in 4% paraformaldehyde in 0.1 M PB (pH 7.4),and stored in 20% sucrose in 0.1 M PB (pH 7.4) until used. Serialsections (20 μm thick) were then cut on a cryostat. Endogenousperoxidase was inactivated by immersing the sections in a solution of0.3% hydrogen peroxide in 10% methanol/0.1M PBS for 30 min. To blocknonspecific staining, sections were incubated in 5% normal horse serumin 0.1 M PBS containing 0.3% Triton X-100 for 1 hr. After blocking, thesections were incubated overnight with the following antibodies (mouseor goat anti-rat) (dilutions in parentheses): against the majorhistocompatibility complex (MHC) class II (Ia) antigen (OX 6, Serotec,Raleigh, N.C., USA, 1:100), against TNF (YC032, Yanaihara Institute,Fujinomiya, Shizuoka, Japan 1:800) and against E-selectin (R and Dsystems, Minneapolis, Minn., USA 50 μg/ml). The sections weresubsequently incubated with a biotinylated anti-mouse IgG or abiotinylated anti-goat IgG (Vector Laboratories, Burlingame, Calif.,USA, 1:200) for 1 hr, and then incubated with an avidin-biotinperoxidase complex solution (Vector Laboratories, Burlingame, Calif.,USA, 1:100) for 1 hr. After each incubation other than that for blockingnonspecific staining, the sections were rinsed for 15 min with 0.1 M PBScontaining 0.3% Triton X-100. Finally, the immunoreaction products werevisualized with diaminobenzidine (DAB kit, Vector Laboratories,Burlingame, Calif., USA). For assessment of nonspecific staining,primary antibodies were replaced with normal mouse or goat IgG. Wecounted the numerical density of the MHC class II (Ia) antigenimmunopositive microglia/macrophages in a 0.75 mm² area in the corpuscallosum and the number of TNF or E-selectin immunopositive vessels inthe total area of the corpus callosum in a section at a level of −2.3 mmfrom bregma. To evaluate the hippocampal damage, 20 micrometer-thickfrozen sections including hippocampus were stained with cresyl violet.

Immunoassay: The level of plasma TNF concentration was measured by a RatTNF US ELISA kit (BioSource International, Camarillo, Calif., USA)following the manufacturer's instructions. The O.D. values (450 nm) weremeasured by SpectraMax M5 (Molecular Devices, Sunnyvale, Calif., USA)and the concentration of the plasma TNF was calculated.

Statistical analysis: Data are represented as mean±SD. Differences inthe mortality rates between groups were determined by Fisher's exactprobability test. Differences in the change of ear thickness between thegroups were determined by unpaired Student's t-test. Differences inproportions of the T maze spontaneous alternation among each of thethree groups were determined by x² test. Differences in thediscrimination index of the object recognition test and the percentagesof correct arm entries on the T-maze left/right discrimination memoryretention test among the groups were determined by repeated measureanalysis of variance (ANOVA) followed by post-hoc testing with Fisher'sprotected least significant difference procedure. Differences in thefiber densities were determined by two-factor ANOVA followed by Fisher'sprotected least significant difference post-hoc testing. Differences inthe numerical densities of either the MHC class II antigenimmunoreactive microglia/macrophages, the TNF immunoreactive vessels orE-selectin immunoreactive vessels and in the level of plasma TNFconcentration were determined by one-factor ANOVA followed by Fisher'sprotected least significant difference post-hoc testing. To evaluate thepossible effect of optic nerve damage on the object recognition test, aPearson correlation coefficient was calculated between the fiber densityof the optic nerve and discrimination index in the E-selectin treatedanimals. p<0.05 was considered significant.

Results

Mortality rates: None of the sham-operated animals died. Of the 11animals that received E-selectin, 2 animals (18.2%) died within 7 daysafter surgery, one animal (9.1%) died by the anesthesia for the nasalinstillation of E-selectin at 9 weeks after surgery. Of the 11 animalsthat received PBS, 4 animals (27.3%) died within 7 days after surgery.There was no significant difference in the mortality rates between theE-selectin and PBS groups.

Cerebral blood flow (CBF) without E-Selectin tolerization: Cerebralblood flow (CBF) was 30 to 50% of the control several days afterligation. The CBF decreased to values ranging from 40 to 80% of controlover a prolonged period (1 week-1 month).

Delayed-type hypersensitivity after E-selectin treatment: A singlecourse of tolerization with E-selectin significantly suppressed the earswelling in the delayed-type hypersensitivity study (p=0.0255). FIG. 2shows that rats treated with E-selectin had an ear thickness of slightlyless than 0.05 mm whereas control rats that received only PBS had an earthickness of almost 0.07 mm. These data indicate that rats treated withE-selectin became tolerized to later E-selectin administration in theear flap and therefore did not exhibit as much inflammation andswelling.

Behavioral Assessment

As described in more detail below, tolerization with E-selectinsignificantly improved the learning and memory impairment in the objectrecognition test (FIG. 3), T-maze memory retention (FIG. 5) and theability to handle changes in the T-maze (FIG. 4), compared with thecontrol group of rats that received only PBS.

Neurological impairment without E-Selectin tolerization: Gaitperformance declined over time in comparison with baseline. At 60 and 90days, bilateral common carotid artery occlusion rats showed decreasedperformances on object recognition and T maze spontaneous alternationtest in comparison with sham-operated rats.

Object recognition test: There were no significant differences in thediscrimination index among the E-selectin, PBS and sham groups beforesurgery (baseline).

After surgery, the PBS group developed a reduced discrimination index.In contrast, the discrimination indices of the E-selectin and shamgroups were maintained at the same baseline levels throughout theexperiment. The discrimination indices of the PBS group weresignificantly decreased as compared with the E-selectin and sham groups(p=0.0005, p=0.0059 respectively). There were no significant differencesin the discrimination index between the E-selectin and the sham groups(p=0.7397). Thus, induction and maintenance of mucosal tolerance toE-selectin protected against the decrease in discrimination observed inthe PBS group. (FIG. 3).

T-maze spontaneous alternation: There were no significant differences inthe percentage of spontaneously alternating rats among the E-selectin,PBS and sham groups before surgery and at 2 and 6 weeks after surgery.

However, by 10 weeks after surgery, the percentage of spontaneouslyalternating rats in the PBS group was significantly decreased comparedwith the E-selectin group (p<0.05). Thus mucosal tolerization toE-selectin protected against loss of the spontaneous alternationtendency seen in PBS tolerized rats. (FIG. 4).

T-Maze Left/Right Discrimination Memory Retention

Two weeks after surgery: The numbers of correct arm entries did notdiffer among the E-selectin, PBS and sham groups tested 1 day after thetraining session. The percentages of correct arm entries were greaterthan 95%. However, in the PBS group, the number of correct arm entriesdecreased over time, and the percentage of correct entries between 3 and14 days after the training session were diminished to 50 to 60%, whichis close to a random choice level. This suggests that animals in thisgroup had lost their left/right discrimination memory. The decrease inthe number of correct arm entries was less prominent in the E-selectingroup, and rats treated with E-selectin had a statisticallysignificantly higher number of correct entries than the PBS-treatedanimals by repeated measure ANOVA (p<0.0001). In contrast with the PBSand E-selectin groups, the sham group retained their left/rightdiscrimination memory at the same level throughout the experiment. Thedifference between the sham and the PBS groups was statisticallysignificant (p<0.0001), and the difference between the sham and theE-selectin groups was also statistically significant by repeated measureANOVA (p=0.0040) (FIG. 5A).

Six weeks after surgery: The E-selectin group had a higher number ofcorrect entries than the PBS group by repeated measure ANOVA (p=0.008)(FIG. 5B). However, the difference between sham and E-selectin groupswas still statistically significant by repeated measure ANOVA (p=0.0030)(FIG. 5B). The difference between sham and PBS groups was alsostatistically significant (p<0.0001). Hence, at six weeks, E-selectintolerization had not completely ameliorated the effects of carotidligation.

Ten weeks after surgery: In contrast with 2 and 6 weeks after surgery,the sham and the E-selectin groups both retained their left/rightdiscrimination memory throughout the experiment, and there were nosignificant differences in the number of correct entries between thesetwo groups by repeated measure ANOVA (p=0.6256). The E-selectin and shamgroups had a higher number of correct entries than the PBS group byrepeated measure ANOVA (p=0.0003, and p=0.0026, respectively) (FIG. 5C).Thus, mucosal tolerance to E-selectin led recovery of the spatialreference memory in left/right discrimination tasks.

Histopathology: In the sham-operated animals, there was no detectablerarefaction in the white matter. However, rarefaction of the whitematter was observed in the corpus callosum, in caudoputamen traversingfiber bundles and in the optic nerve in the PBS-treated rats. For thesestudies, luxol fast blue stain and immunocytochemistry with a cocktailof antibodies directed against nonphosphorylated neurofilaments (SMI311) were used. The severity of the rarefaction was markedly attenuatedin the animals treated with E-selectin (FIGS. 6, 7). In particular, thefiber densities in the E-selectin-treated animals were significantlyhigher than those in the PBS-treated group by two-factor ANOVA(p<0.0001). Moreover, there was no significant difference in the fiberdensities between sham and E-selectin groups (p=0.2026) (FIG. 8). Thus,E-selectin mucosal tolerization had a protective effect against whitematter rarefaction induced by protracted hypoperfusion.

The Pearson correlation coefficient between the fiber density of theoptic nerve and the discrimination index in the E-selectin treatedanimals was minus 0.470. This correlation was not significantlydifferent from 0 (p=0.2537). A few dark neurons were detected in theunilateral hippocampus of the three E-selectin-treated (27.3%), threePBS-treated animals (27.3%) and one sham-operated animal (25%). Therewere no obvious differences in the number of the dark neurons amongthree groups (data not shown).

In the white matter of the sham-operated animals, there was positiveimmunostaining for the MHC class II (Ia) antigen in only a few glialcells. However, the brains of the PBS-treated animals showed an increasein the number of microglia/macrophages that were immunolabeled for theMHC class II (Ia) antigen. These microglia and macrophages were observedin the white matter within the corpus callosum and caudoputamen. Incontrast, in E-selectin-treated rats, the number ofmicroglia/macrophages positively immunolabeled for MHC class II antigentended to correlate with a tendency towards a decrease in the whitematter lesions as compared to PBS-treated animals (FIG. 9). However,there were no significant differences among E-selectin-treated group,sham-operated group and PBS-treated group (FIG. 10).

While TNF-α was prominently expressed in endothelial cells in bloodvessels of the white matter, such TNF-α expression was markedlyattenuated in E-selectin-tolerized and sham-operated animals (FIG. 12).The TNF immunoreactive vessels were significantly (p=0.0016) decreasedin number in the E-selectin-treated groups as compared to thePBS-treated group. In contrast, there were no significant differences inthe number of TNF immunoreactive vessels between the sham and E-selectingroups (p=0.5725) (FIG. 13).

E-selectin was expressed in endothelial cells of vessels in the brainsof the PBS-treated animals. The E-selectin immunoreactive vessels weredecreased in number in the E-selectin-treated group as compared to thePBS-treated group (FIG. 14). The E-selectin-treated animals exhibited asignificant reduction (p=0.0001) in the number of E-selectinimmunopositive vessels as compared to the PBS-treated group. Incontrast, there were no significant differences in the number ofE-selectin immunoreactive vessels between sham and the E-selectin groups(p=0.5537) (FIG. 15). Thus, the mucosal tolerance to E-selectin had asuppressive effect against the activation of vessels in the braininduced by protracted hypoperfusion.

Immunoassay: The sham group had a statistically significantly lowerlevel of plasma TNF than the E-selectin and PBS groups by one-factorANOVA (p<0.05). However, there were no significant differences on thelevel of plasma TNF between the E-selectin and PBS groups.

The results provided above illustrate the protective effect of mucosaltolerance to E-selectin against histological damage and functionalimpairments that develops during protracted cerebral hypoperfusioninduced by the permanent occlusion of both common carotid arteries.

Because the severity of the damage in the optic nerve was attenuated inthe E-selectin-treated group as compared to the PBS-treated group, thepotential effect of differential visual acuity on behavioral tests suchas the object recognition test should be considered. In this study, thediscrimination ability preserved by E-selectin treatment was notcorrelated with the degree of protection from fiber loss in the opticnerve. One explanation for this discrepancy is that humans primarilybase their choices on a memory of visual the properties of the sampleobject. In contrast, when rats explore an object, they sniff it, palpateit with vibrissae, and look at it. In the rodents, differentialexploration of familiar objects and novel objects reflects to someextent their memory for olfactory and tactile properties of the sampleobject, although visual properties may also be remembered and contributeto discrimination.

There were apparent differences in the protective effects conferred byE-selectin tolerization on T maze left/right discrimination memory whentested at the 2, 6 and 10 week time points. In contrast to 10 weeksafter surgery, at 2 and 6 weeks after surgery the E-selectin group didnot retain their left/right discrimination memory. The impairment in theleft/right discrimination memory at 2 and 6 weeks after surgery mighthave been caused by a decrease of cerebral blood flow that impairedfunction without permanent cortical white matter damage. Cerebral bloodflow in this model remains reduced over a prolonged period and graduallyrecovers to control levels by 8 weeks (Otori T, Cerebrovasc. Dis. 6(suppl): 71 (1996)).

In the present study, plasma TNF level was increased in the ischemicgroups (PBS and E-selectin), as compared with the sham-operated groupeven 90 days after surgery. Since the expression of E-selectin isinduced in response to TNF, these findings suggest that the endothelialactivation and E-selectin induction could persist for a prolonged periodunder conditions of protracted hypoperfusion. Mucosal tolerance can beachieved through different mechanisms, including clonal anergy/deletionof antigen-reactive T cells, and active tolerance with induction ofregulatory T cells (Faria A M, Adv Immunol. 73:153-264 (1999)). Clonalanergy/deletion can be induced by a single feeding of very high-doseantigen (Chen Y., Nature. 376(6536):177-180 (1995)) and the productionof regulatory T cells occurs after repetitive administration of low-doseantigen (Groux H., Nature 389:737-742 (1997); Chen Y., Science265:1237-1240 (1994)). Lymphocytes that are tolerized to an antigen andhave become antigen-specific regulatory T-cells tend to migrate to thelocale of the protein molecule to which they have been primed. In thatlocation, they release immunomodulatory cytokines, such as TGF-β andIL-10 that counteract the effect of pro-inflammatory cytokines includingTNF-α and suppress inflammation and immune responses after ischemia(Pang L., Stroke. 2001; 32:544-552 (2001), Hallenbeck J. M., Trends inImmunology 26:550-556 (2005)). Since local release of immunological andinflammatory mediators contributes to local vessel activation, localimmunosuppression targeted to activating blood vessel segments couldprotect against local impairment of microcirculatory perfusion. In thisstudy, the number of TNF immunopositive vessels and E-selectinimmunopositive vessels were significantly decreased in the E-selectintolerized group, compared to the PBS control group. But the plasma TNFlevel was not significantly decreased in the E-selectin group ascompared with PBS group. These results indicate that local vesselactivation and local TNF production was suppressed by the mucosaltolerance to E-selectin in a setting of undiminished systemic TNFproduction. TNF expressed by endothelium has proinflammatory andprocoagulant effects on endothelium (Pober J S, Physiol Rev. 70:427-451(1990), Hallenbeck J M. Nat. Med. 8:1363-1368 (2002)). E-selectinappears to function by suppressing local vessel activation and thesurrounding immunological and inflammatory processes rather than bysystemic immunosuppression.

Other mechanisms may also contribute to the protective effect in thepresent study. White matter injury involves glial cells, which areabundant in white matter (Goldberg M, Stroke 34:330-332 (2003)). In thismodel, microglial activation with expression of MHC class II antigenswas detected preferentially in the white matter (Wakita H, ActaNeuropathol. (Berl) 87: 484-492 (1994); Farkas E., Acta Neuropathol(Berl). 108:57-64 (2004), Schmidt-Kastner R., Brain Res. 1052:28-39(2005)), and pharmacological suppression of these activated microgliahas resulted in an attenuation of the white matter lesions (Wakita H,Stroke 26:1415-1422 (1995); Wakita H, Brain Res. 792:105-113 (1998);Wakita H, Neuroreport 14:1461-1465 (1999), Wakita H., Brain Res.992:53-59 (2003)).

Since TGF-β and IL-10 inhibit the activation of microglia (Suzumura A,J. Immunol. 151:2150-2158 (1993), Frei K, J. Immunol. 152:2720-2728(1994)), mucosal tolerization to E-selectin suppresses the activatedmicroglia through local production of these cytokines by the regulatoryT cells. The number of MHC class II positive activatedmicroglia/macrophages in the white matter showed a trend towardsuppression in the E-selectin group as compared to the PBS group.Activated microglia may enhance a variety of inflammatory responses(Morioka T, J. Cereb. Blood Flow Metab. 1991; 11:966-973 (1991); WakitaH, Acta Neuropathol. (Berl) 87: 484-492 (1994); Gehrmann J, Brain Res.Rev. 20:269-287 (1995)). Microglia are the major source ofpro-inflammatory cytokines including IL-1 and TNF, which may induce theexpression of E-selectin in the ischemic cerebral vasculature. Thesuppression of the microglia may inhibit both local vessel activationand the expression of E-selectin. Activated microglia also release anarray of cytotoxic substances that include other pro-inflammatorycytokines, prostanoids, proteases, reactive oxygen radicals and nitrogenintermediates. The protective effect may be mediated by suppressing therelease of these cytotoxic substances as well. The net effect decreasesinflammation and preserves vessel integrity.

In conclusion, the present study demonstrates the protective effect ofmucosal tolerance to E-selectin against ischemic cerebrovascular whitematter damage and memory impairment during protracted cerebralhypoperfusion. These results support a new therapeutic strategy thatinvolves mucosal tolerization to E-selectin to protect againstsubcortical ischemic vascular cognitive impairment on a long-term basis.

Example 5 E-Selectin Sequences Employed

For early experiments, a human E-selectin polypeptide with SEQ ID NO: 30was made by recombinant procedures using a pNVAX1002 expression vector.This SEQ ID NO: 30 sequence is shown below.

  1 MGWSWIFLFL LSGTASVHSW SYNTSTEAMT YDEASAYCQQ 41 RYTHLVAIQN KEEIEYLNSI LSYSPSYYWI GIRKVNNVWV 81 WVGTQKPLTE EAKNWAPGEP NNRQKDEDCV EIYIKREKDV121 GMWNDERCSK KKLALCYTAA CTNTSCSGHG ECVETINNYT161 CKCDPGFSGL KCEQIVNCTA LESPEHGSLV CSHPLGNFSY201 NSSCSISCDR GYLPSSMETM QCMSSGEWSA PIPACNVVEC241 DAVTNPANGF VECFQNPGSF PWNTTCTFDC EEGFELMGAQ281 SLQCTSSGNW DNEKPTCKAV TGGASTRAAE QKLISEEDLN 321 GTRSGHHHHH HThis SEQ ID NO: 30 sequence has a signal sequence (MGWSWIFLFL LSGTASVHS(SEQ ID NO: 27)), which is cleaved during recombinant production and isnot present in the purified product. The SEQ ID NO: 30 E-selectinsequence also has a histidine tag sequence (GGASTRAAEQKLISEEDLNGTRSGHHHHHH (SEQ ID NO: 29)), which can facilitate isolation anddetection of the E-selectin. Upon removal of the MGWSWIFLFL LSGTASVHS(SEQ ID NO: 27) signal sequence a polypeptide with the followingE-selectin polypeptide with SEQ ID NO: 31 is generated.

  1                     W SYNTSTEAMT YDEASAYCQQ 41 RYTHLVAIQN KEEIEYLNSI LSYSPSYYWI GIRKVNNVWV 81 WVGTQKPLTE EAKNWAPGEP NNRQKDEDCV EIYIKREKDV121 GMWNDERCSK KKLALCYTAA CTNTSCSGHG ECVETINNYT161 CKCDPGFSGL KCEQIVNCTA LESPEHGSLV CSHPLGNFSY201 NSSCSISCDR GYLPSSMETM QCMSSGEWSA PIPACNVVEC241 DAVTNPANGF VECFQNPGSF PWNTTCTFDC EEGFELMGAQ281 SLQCTSSGNW DNEKPTCKAV TGGASTRAAE QKLISEEDLN 321 GTRSGHHHHH H

For somewhat later experiments, a human E-selectin polypeptide with SEQID NO: 32 was made by recombinant procedures using a pNVAX1037expression vector. This SEQ ID NO: 32 sequence is shown below.

  1 MGWSWIFLFL LSGTASVHSW SYNTSTEAMT YDEASAYCQQ 41 RYTHLVAIQN KEEIEYLNSI LSYSPSYYWI GIRKVNNVWV 81 WVGTQKPLTE EAKNWAPGEP NNRQKDEDCV EIYIKREKDV121 GMWNDERCSK KKLALCYTAA CTNTSCSGHG ECVETINNYT161 CKCDPGFSGL KCEQIVNCTA LESPEHGSLV CSHPLGNFSY201 NSSCSISCDR GYLPSSMETM QCMSSGEWSA PIPACNVVEC241 DAVTNPANGF VECFQNPGSF PWNTTCTFDC EEGFELMGAQ281 SLQCTSSGNW DNEKPTCKAV TThis SEQ ID NO: 32 sequence has a signal sequence (MGWSWIFLFL LSGTASVHS(SEQ ID NO: 27)) but no histidine tag sequence. As indicated above, theSEQ ID NO: 27 signal sequence is cleaved during recombinant productionand is not present in the purified product. Upon removal of the SEQ IDNO: 27 signal sequence, this E-selectin polypeptide has SEQ ID NO: 8.

For more recent experiments, a mouse E-selectin polypeptide with SEQ IDNO: 33 was made by recombinant procedures using a pNVAX1076 expressionvector. This SEQ ID NO: 33 sequence is shown below.

  1 MPLYKLLNVL WLVAVSNAIW YYNASSELMT YDEASAYCQR 41 DYTHLVAIQN KEEINYLNSN LKHSPSYYWI GIRKVNNVWI 81 WVGTGKPLTE EAQNWAPGEP NNKQRNEDCV EIYIQRTKDS121 GMWNDERCNK KKLALCYTAS CTNASCSGHG ECIETINSYT161 CKCHPGFLGP NCEQAVTCKP QEHPDYGSLN CSHPFGPFSY201 NSSCSFGCKR GYLPSSMETT VRCTSSGEWS APAPACHVVE241 CEALTHPAHG IRKCSSNPGS YPWNTTCTFD CVEGYRRVGA281 QNLQCTSSGI WDNETPSCKA VTThis SEQ ID NO: 33 sequence has an N-terminal signal sequence(MPLYKLLNVLWLVAVSNAI (SEQ ID NO: 28)), which is cleaved and lost duringrecombinant production of the E-selectin product. Upon removal of theSEQ ID NO: 28 signal sequence, this E-selectin polypeptide has SEQ IDNO: 19.

Recent experiments have also employed a mouse E-selectin polypeptidewith SEQ ID NO: 18 was made by recombinant procedures using a pNVAX1189expression vector. This SEQ ID NO: 18 sequence is shown below.

  1 MGWSWIFLFL LSGTASVHSW YYNASSELMT YDEASAYCQR 41 DYTHLVAIQN KEEINYLNSN LKHSPSYYWI GIRKVNNVWI 81 WVGTGKPLTE EAQNWAPGEP NNKQRNEDCV EIYIQRTKDS121 GMWNDERCNK KKLALCYTAS CTNASCSGHG ECIETINSYT161 CKCHPGFLGP NCEQAVTCKP QEHPDYGSLN CSHPFGPFSY201 NSSCSFGCKR GYLPSSMETT VRCTSSGEWS APAPACHVVE241 CEALTHPAHG IRKCSSNPGS YPWNTTCTFD CVEGYRRVGA281 QNLQCTSSGI WDNETPSCKA VTThis SEQ ID NO: 18 sequence has a signal sequence (MGWSWIFLFL LSGTASVHS(SEQ ID NO: 27)), which is cleaved during recombinant production and isnot present in the purified product. Upon removal of the SEQ ID NO: 27signal sequence, this E-selectin polypeptide has SEQ ID NO: 19.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “a host cell” includes a plurality (forexample, a culture or population) of such host cells, and so forth.Under no circumstances may the patent be interpreted to be limited tothe specific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. An isolated E-selectin polypeptide consisting of the lectin, EGF, CR1and CR2 domains.
 2. The polypeptide of claim 1, wherein the polypeptideconsists of the amino acid sequence of SEQ ID NO:
 8. 3. A compositioncomprising the polypeptide of claim 1 and a pharmaceutically acceptablecarrier.
 4. An isolated nucleic acid molecule encoding the polypeptideof claim
 1. 5. A vector comprising the nucleic acid molecule of claim 4.6. A method of treating an inflammation mediated disease or condition inan individual in need thereof by inducing mucosal tolerance to a solubleE-selectin polypeptide, comprising administering to the individualmultiple low doses of E-selectin through nasal administration, whereinthe E-selectin polypeptide consists of the polypeptide of claim
 1. 7. Anisolated E-selectin polypeptide consisting of an amino-terminal signalsequence and the lectin, EGF, CR1 and CR2 domains.
 8. The polypeptide ofclaim 7, wherein the signal sequence comprises SEQ ID NO: 27 or SEQ IDNO:
 28. 9. The polypeptide of claim 7, wherein the polypeptide consistsof the amino acid sequence of SEQ ID NO:
 32. 10. A compositioncomprising the polypeptide of claim 7 and a pharmaceutically acceptablecarrier.
 11. An isolated nucleic acid molecule encoding the polypeptideof claim
 7. 12. A vector comprising the nucleic acid molecule of claim11.
 13. A method of treating an inflammation mediated disease orcondition in an individual in need thereof by inducing mucosal toleranceto a soluble E-selectin polypeptide, comprising administering to theindividual multiple low doses of E-selectin through nasaladministration, wherein the E-selectin polypeptide consists of thepolypeptide of claim 7.